Stabilized liquid anti-RSV antibody formulations

ABSTRACT

The present invention provides liquid formulations of SYNAGIS® or an antigen-binding fragment thereof that immunospecifically bind to a respiratory syncytial virus (RSV) antigen, which formulations exhibit stability, low to undetectable levels of aggregation, and very little to no loss of the biological activities of SYNAGIS® or an antigen-binding fragment thereof, even during long periods of storage. In particular, the present invention provides liquid formulations of SYNAGIS® or an antigen-binding fragment thereof which immunospecifically binds to a RSV antigen, which formulations are substantially free of surfactant, inorganic salts, and/or other common excipients. Furthermore, the invention provides method of preventing, treating or ameliorating symptoms associated with RSV infection utilizing liquid formulations of the present invention.

This application is entitled to and claims priority benefit to U.S.provisional application Ser. No. 60/388,921 filed Jun. 14, 2002, whichis incorporated herein by reference in its entirety.

1. INTRODUCTION

The present invention relates to liquid formulations of SYNAGIS®(palivizumab) or an antigen-binding fragment thereof, which formulationsexhibit stability, low to undetectable levels of antibody fragmentation,low to undetectable levels of aggregation, and very little to no loss ofthe biological activity (e.g., therapeutic efficacy) SYNAGIS® or anantigen-binding fragment thereof, even during or after long periods ofstorage. In particular, the present invention relates to liquidformulations of SYNAGIS® or an antigen-binding fragment thereof, whichformulations are substantially free of surfactant and/or inorganicsalts. The present invention also relates to methods of preventing,treating or ameliorating symptoms associated with a respiratorysyncytial virus (RSV) infection utilizing liquid formulations ofSYNAGIS® or an antigen binding fragment thereof.

2. BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is the leading cause of serious lowerrespiratory tract disease in infants and children (Feigen et al., eds.,1987, In: Textbook of Pediatric Infectious Diseases, WB Saunders,Philadelphia at pages 1653–1675; New Vaccine Development, EstablishingPriorities, Vol. 1, 1985, National Academy Press, Washington DC at pages397–409; and Ruuskanen et al., 1993, Curr. Probl. Pediatr. 23:50–79).The yearly epidemic nature of RSV infection is evident worldwide, butthe incidence and severity of RSV disease in a given season vary byregion (Hall, C. B., 1993, Contemp. Pediatr. 10:92–110). In temperateregions of the northern hemisphere, it usually begins in late fall andends in late spring (Hall, C. B., 1995, In: Mandell G. L., Bernnett J.E., Dolin R., eds., 1995, Principles and Practice of InfectionsDiseases. 4th ed., Churchill Livingstone, New York at pages 1501–1519).It is estimated that RSV illness results in 90,000 hospitalizations andcauses 4,500 deaths annually in the United States. Primary RSV infectionoccurs most often in children from 6 weeks to 2 years of age anduncommonly in the first 4 weeks of life during nosocomial epidemics(Hall et al., 1979, New Engl. J. Med. 300:393–396). RSV is estimated tocause as much as 75% of all childhood bronchiolitis and up to 40% of allpediatric pneumonias (Cunningham, C. K. et al., 1991, Pediatrics88:527–532). Children at increased risk from RSV infection includepreterm infants (Hall et al., 1979, New Engl. J. Med. 300:393–396) andchildren with bronchopulmonary dysplasia (Groothuis et al., 1988,Pediatrics 82:199–203), congenital heart disease (MacDonald et al., NewEngl. J. Med. 307:397–400), congenital or acquired immunodeficiency(Ogra et al., 1988, Pediatr. Infect. Dis. J. 7:246–249; and Pohl et al.,1992, J. Infect. Dis. 165:166–169), and cystic fibrosis (Abman et al.,1988, J. Pediatr. 113:826–830). The fatality rate in infants with heartor lung disease who are hospitalized with RSV infection is 3%–4% (Navaset al., 1992, J. Pediatr. 121:348–354).

RSV infects adults as well as infants and children. In healthy adults,RSV causes predominantly upper respiratory tract disease. It hasrecently become evident that some adults, especially the elderly, havesymptomatic RSV infections more frequently than had been previouslyreported (Evans, A. S., eds., 1989, Viral Infections of Humans.Epidemiology and Control, 3^(rd) ed., Plenum Medical Book, New York atpages 525–544). Several epidemics also have been reported among nursinghome patients and institutionalized young adults (Falsey, A. R., 1991,Infect. Control Hosp. Epidemiol. 12:602–608; and Garvie et al., 1980,Br. Med. J. 281:1253–1254). Finally, RSV may cause serious disease inimmunosuppressed persons, particularly bone marrow transplant patients(Hertz et al., 1989, Medicine 68:269–281).

Treatment options for established RSV disease are limited. Severe RSVdisease of the lower respiratory tract often requires considerablesupportive care, including administration of humidified oxygen andrespiratory assistance (Fields et al., eds, 1990, Fields Virology,2^(nd) ed., Vol. 1, Raven Press, New York at pages 1045–1072). The onlydrug approved for treatment of infection is the antiviral agentribavirin (American Academy of Pediatrics Committee on InfectiousDiseases, 1993, Pediatrics 92:501–504). It has been shown to beeffective in the treatment of RSV pneumonia and bronchiolitis, modifyingthe course of severe RSV disease in immunocompetent children (Smith etal., 1991, New Engl. J. Med. 325:24–29). However, ribavirin has a numberof limitations including high cost, need for prolonged aerosoladministration and potential risk to pregnant women as well as toexposed health care personnel. The American Academy of PediatricsCommittee on Infectious Diseases revised their recommendation for use ofribavirin. The current recommendation is that the decision to useribavirin should be based on the particular clinical circumstances andphysician's experience (American Academy of Pediatrics. Summaries ofInfectious Diseases. In: Pickering L. K., ed., 2000 Red Book: Report ofthe Committee on Infectious Diseases. 25th ed., Elk Grove Village, Ill.,American Academy of Pediatrics, 2000, pp. 483–487).

While a vaccine might prevent RSV infection, no vaccine is yet licensedfor this indication. A major obstacle to vaccine development is safety.A formalin-inactivated vaccine, though immunogenic, unexpectedly causeda higher and more severe incidence of lower respiratory tract diseasedue to RSV in immunized infants than in infants immunized with asimilarly prepared trivalent parainfluenza vaccine (Kim et al., 1969,Am. J. Epidemiol. 89:422–434; and Kapikian et al., 1969, Am. J.Epidemiol. 89:405–421). Several candidate RSV vaccines have beenabandoned and others are under development (Murphy et al., 1994, VirusRes. 32:13–36), but even if safety issues are resolved, vaccine efficacymust also be improved. A number of problems remain to be solved.Immunization would be required in the immediate neonatal period sincethe peak incidence of lower respiratory tract disease occurs at 2–5months of age. The immaturity of the neonatal immune response togetherwith high titers of maternally acquired RSV antibody may be expected toreduce vaccine immunogenicity in the neonatal period (Murphy et al.,1988, J. Virol. 62:3907–3910; and Murphy et al., 1991, Vaccine9:185–189). Finally, primary RSV infection and disease do not protectwell against subsequent RSV disease (Henderson et al., 1979, New Engl.J. Med. 300:530–534).

Currently, the only approved approach to prophylaxis of RSV disease ispassive immunization. Initial evidence suggesting a protective role forIgG was obtained from observations involving maternal antibody inferrets (Prince, G. A., Ph.D. diss., University of California, LosAngeles, 1975) and humans (Lambrecht et al, 1976, J. Infect. Dis.134:211–217; and Glezen et al., 1981, J. Pediatr. 98:708–715). Hemminget al. (Morell et al., eds., 1986, Clinical Use of IntravenousImmunoglobulins, Academic Press, London at pages 285–294) recognized thepossible utility of RSV antibody in the treatment or prevention of RSVinfection during studies involving the pharmacokinetics of anintravenous immune globulin (IVIG) in newborns suspected of havingneonatal sepsis. They noted that one infant, whose respiratorysecretions yielded RSV, recovered rapidly after IVIG infusion.Subsequent analysis of the IVIG lot revealed an unusually high titer ofRSV neutralizing antibody. This same group of investigators thenexamined the ability of hyperimmune serum or immune globulin, enrichedfor RSV neutralizing antibody, to protect cotton rats and primatesagainst RSV infection (Prince et al., 1985, Virus Res. 3:193–206; Princeet al., 1990, J. Virol. 64:3091–3092; Hemming et al., 1985, J. Infect.Dis. 152:1083–1087; Prince et al., 1983, Infect. Immun. 42:81–87; andPrince et al., 1985, J. Virol. 55:517–520). Results of these studiessuggested that RSV neutralizing antibody given prophylacticallyinhibited respiratory tract replication of RSV in cotton rats. Whengiven therapeutically, RSV antibody reduced pulmonary viral replicationboth in cotton rats and in a nonhuman primate model. Furthermore,passive infusion of immune serum or immune globulin did not produceenhanced pulmonary pathology in cotton rats subsequently challenged withRSV.

A humanized antibody directed to an epitope in the A antigenic site ofthe F protein of RSV, SYNAGIS®, comprising variable heavy (VH)complementarity determining regions (CDRs) having the amino acidsequences of SEQ ID NO:1–3 and variable light (VL) CDRs having the aminoacid sequences of SEQ ID NO:4–6, is approved for intramuscularadministration to pediatric patients for prevention of serious lowerrespiratory tract disease caused by RSV at recommended monthly doses of15 mg/kg of body weight throughout the RSV season (November throughApril in the northern hemisphere). SYNAGIS® is a composite of human(95%) and murine (5%) antibody sequences. See, Johnson et al., 1997, J.Infect. Diseases 176:1215-1224 and U.S. Pat. No. 5,824,307, the entirecontents of which are incorporated herein by reference. The human heavychain sequence was derived from the constant domains of human IgG₁ andthe variable framework regions of the VH genes of Cor (Press et al.,1970, Biochem. J. 117:641–660) and Cess (Takashi et al., 1984, Proc.Natl. Acad. Sci. USA 81:194–198). The human light chain sequence wasderived from the constant domain of C6 and the variable frameworkregions of the VL gene K104 with J6-4 (Bentley et al., 1980, Nature288:5194–5198). The murine sequences were derived from a murinemonoclonal antibody, Mab 1129 (Beeler et al., 1989, J. Virology63:2941–2950), in a process which involved the grafting of the murinecomplementarity determining regions into the human antibody frameworks.

SYNAGIS® has high specific activity against RSV in vitro (approximately50–100 times that of RESPIGAM® (respiratory syncytial virus immuneglobulin, human) and is known to neutralize a broad range of RSVisolates. Since it is not derived from human plasma, prophylactictreatment with SYNAGIS® does not carry potential risk of transmission ofblood borne pathogens.

SYNAGIS® was initially formulated as a liquid for IV use, at aconcentration of 10 mg/ml SYNAGIS® in phosphate buffered saline. Alyophilized formulation of SYNAGIS®, which allows a higher concentration(100 mg/ml after reconstitution, in 50 mM histidine and 3.2 mM glycinebuffer with 6% (w/v) mannitol at pH 6.0) of the antibody than thisinitial liquid formulation, was produced later to allow intramuscularuse. The lyophilized formulation of SYNAGIS® is prepared by lyophilizingSYNAGIS® at 54 mg/ml in an aqueous solution containing 25 mM histidine,1.6 mM glycine, and 3% (w/v) mannitol at pH 6.0. The initial liquidformulation in PBS and the lyophilized formulation of SYNAGIS® have beentested in phase I clinical studies in healthy adults. The lyophilizedformulation was tested in phase I through phase IV studies in pediatricpatients. SYNAGIS®, at doses of 15 mg/kg to 30 mg/kg for adults is foundto be well tolerated, and 15 mg/kg for children is found to be safe andefficacious for RSV prophylaxis. The lyophilized formulation wasapproved in 1998 by the FDA for use in the prevention of serious lowerrespiratory tract disease caused by RSV in children at high risk of RSVdisease.

However, the lyophilized formulation has a number of limitations,including a prolonged process for lyophilization and resulting high costfor manufacturing. In addition, the lyophilized formulation has to bereconstituted aseptically and accurately by healthcare practitionersprior to administering to patients. The reconstitution step itselfrequires certain specific procedures: (1) a sterile diluent (i.e., wateror 5% dextrose in water for intravenous administration and water forintramuscular administration) is added to the vial containinglyophilized SYNAGIS®, slowly and aspetically, and the vial must beswirled very gently for 30 seconds to avoid foaming; (2) thereconstituted SYNAGIS® needs to stand at room temperature for a minimumof 20 minutes until the solution clarifies; and (3) the reconstitutedpreparation must be administered within six (6) hours after thereconstitution. Such reconstitution procedure is cumbersome and the timelimitation after the reconstitution can cause a great inconvenience inadministering the formulation to patients, leading to significant waste,if not reconstituted properly or if the reconstituted dose is not usedwithin six (6) hours and must be discarded.

Thus, a need exists for a liquid formulation of SYNAGIS® at aconcentration comparable to or higher than the reconstituted lyophilizedformulation so that there is no need to reconstitute the formulationprior to administration. This allows health care practitioners muchquicker and easier administration of SYNAGIS® to a patient.

Prior liquid antibody preparations have short shelf lives and may losebiological activity of the antibodies resulting from chemical andphysical instabilities during the storage. Chemical instability may becaused by deamidation, racemization, hydrolysis, oxidation, betaelimination or disulfide exchange, and physical instability may becaused by antibody denaturation, aggregation, precipitation oradsorption. Among those, aggregation, deamidation and oxidation areknown to be the most common causes of the antibody degradation (Wang etal., 1988, J. of Parenteral Science & Technology 42(Suppl):S4–S26;Cleland et al., 1993, Critical Reviews in Therapeutic Drug CarrierSystems 10(4):307–377). Thus, there is a need for a stable liquidformulation of SYNAGIS® or an antigen-binding fragment thereof effectiveto prevent RSV infection.

3. SUMMARY OF INVENTION

The present invention is based, in part, on the development of highconcentration liquid formulations of SYNAGIS® or an antigen-bindingfragment thereof, which formulations exhibit, in the absence ofsurfactant, inorganic salts, and/or other excipients, stability, low toundetectable levels of antibody fragmentation and/or aggregation, andvery little to no loss of the biological activity(ies) of SYNAGIS® or anantigen-binding fragment thereof during manufacture, preparation,transportation, and storage. The liquid formulations of the presentinvention facilitate the administration of SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management and/or amelioration of a RSV infection or one or moresymptoms thereof. In particular, the liquid formulations of the presentinvention enable a healthcare professional to quickly administer asterile dosage of SYNAGIS® or an antigen-binding fragment thereofwithout having to accurately and aseptically reconstitute the antibodyor antibody fragment prior to administration as required for thelyophilized dosage form. Such liquid formulations of SYNAGIS® can bealso manufactured more easily and cost effectively than the lyophilizedformulation since liquid formulations do not require a prolonged dryingstep, such as lyophilization, freeze-drying, etc. The liquidformulations are made by a process in which the antibody beingformulated is in an aqueous phase throughout the purification andformulation process. Preferably, the liquid formulations are made by aprocess that does not include a drying step, for example, but not by wayof limitation, a lyophilization, freeze-drying, spray-drying, orair-drying step.

The present invention provides liquid formulations of SYNAGIS® or anantigen-binding fragment thereof, substantially free of surfactant,inorganic salts, sugars, and/or other common excipients, saidformulations comprising histidine and a concentration of about 15 mg/mlor higher of SYNAGIS® or an antigen-binding fragment thereof.Optionally, the formulation may further comprise glycine. Alternatively,the formulation of the present invention may further comprise othercommon excipients, such as saccharides, polyols and amino acids,including, but not limited to, arginine, lysine, and methionine. Thepresent invention also provides liquid formulations substantially freeof surfactant, inorganic salts, sugars, and/or other commonly-knownexcipients, said formulation having a pH ranging from about 5.0 to about7.0, preferably about 5.5 to 6.5, more preferably about 5.8 to about6.2, and most preferably about 6.0, and comprising histidine and aconcentration of about 15 mg/ml or higher of SYNAGIS® or anantigen-binding fragment thereof.

The present invention encompasses stable liquid formulations of SYNAGIS®which exhibit low to undetectable levels of antibody aggregation and/orfragmentation with very little to no loss of biological activity(ies) ofSYNAGIS® or an antigen-binding fragment thereof during manufacture,preparation, transportation and long periods of storage. The presentinvention also encompasses stable liquid formulations of modified formsof SYNAGIS® or an antigen-binding fragment thereof that have increasedin vivo half-lives relative to unmodified SYNAGIS® or an antigen-bindingfragment thereof, said formulations exhibiting low to undetectablelevels of antibody aggregation and/or fragmentation, and very little tono loss of biological activity(ies) of SYNAGIS® or an antigen-bindingfragment thereof.

The present invention encompasses liquid formulations of SYNAGIS® or anantigen-binding fragment thereof, said formulations having stability at38°–42° C. as assessed by high performance size exclusion chromatography(HPSEC). The liquid formulations of the present invention exhibitsstability, as assessed by HSPEC, at the temperature ranges of 38° C.–42°C. for at least 60 days (in specific embodiments, not more than 120days), of 20° C.–24° C. for at least 1 year, and of 2° C.–8° C. for atleast 3 years. The present invention also encompasses liquidformulations of SYNAGIS® or an antigen-binding fragment thereof, saidformulations having low to undetectable aggregation as measured byHPSEC. In a preferred embodiment, the liquid formulations of the presentinvention exhibit stability at 38°–42° C. for at least 60 days andexhibit low to undetectable levels of antibody aggregation as measuredby HPSEC, and further, exhibit very little to no loss of biologicalactivity (ies) of SYNAGIS® or an antigen-binding fragment thereofcompared to the reference antibodies as measured by antibody bindingassays such as, e.g., ELISAs.

The present invention provides methods for preparing liquid formulationsof SYNAGIS® or an antigen-binding fragment thereof. The liquidformulations of the present invention are prepared by maintainingSYNAGIS® or an antigen-binding fragment thereof in an aqueous solutionat any time during the preparation. In other words, the liquidformulations are prepared without involving any step of drying SYNAGIS®or an antigen-binding fragment thereof or the formulations themselvesby, for example, lyophilization, vacuum drying, etc.

The present invention provides methods for preparing liquid formulationsof SYNAGIS® or an antigen-binding fragment thereof, said methodscomprising concentrating a fraction of purified SYNAGIS® or anantigen-binding fragment thereof to a final concentration of about 15mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml,about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about100 mg/ml, about 200 mg/ml, about 250 mg/ml, or about 300 mg/ml using asemi-permeable membrane with an appropriate molecular weight (mw) cutoff(e.g., 30 kD cutoff for SYNAGIS® and F(ab′)₂ fragments thereof; and 10kD cutoff for SYNAGIS® fragments such Fab fragments), and diafilteringthe concentrated antibody fraction into the formulation buffer using thesame membrane. The formulation buffer of the present invention compriseshistidine at a concentration ranging from about 1 mM to about 100 mM,about 10 mM to about 50 mM, about 20 mM to about 30 mM, or about 23 mMto about 27 mM, and is most preferably about 25 mM. To obtain anappropriate pH for SYNAGIS® or an antigen-binding fragment thereof, itis preferably that histidine (and glycine, if added) is first dissolvedin water to obtain a buffer solution with higher pH than the desired pHand then the pH is brought down to the desired level by the addition ofHCl. This way, the formation of inorganic salts (e.g., the formation ofNaCl when, e.g., histidine hydrochloride is used as the source ofhistidine and the pH is raised to the desired level by the addition ofNaOH) can be avoided.

The liquid formulations of the present invention may be sterilized bysterile filtration using a 0.2μ or a 0.22μ filter. Sterilized liquidformulations of the present invention may be administered to a subjectto prevent, treat or ameliorate one or more symptoms associated with aRSV infection or a symptom thereof.

The present invention also provides kits comprising the liquidformulations of SYNAGIS® or an antigen-binding fragment thereof for useby, e.g. a healthcare professional. The present invention furtherprovides methods of preventing, treating managing or ameliorating a RSVinfection or one or more symptoms thereof by administering the liquidformulations of the present invention.

3.1 Terminology

As used herein, all liquid formulations of SYNAGIS® and/or fragmentsthereof that immunospecifically bind to a RSV antigen described aboveare collectively referred to as “liquid formulations of the invention,”“SYNAGIS® liquid formulations of the invention,” or “liquid formulationsof SYNAGIS® or an antigen-binding fragment thereof.”

As used herein, the term “cytokine receptor modulator” refers to anagent which modulates the phosphorylation of a cytokine receptor, theactivation of a signal transduction pathway associated with a cytokinereceptor, and/or the expression of a particular protein such as acytokine. Such an agent may directly or indirectly modulate thephosphorylation of a cytokine receptor, the activation of a signaltransduction pathway associated with a cytokine receptor, and/or theexpression of a particular protein such as a cytokine. Thus, examples ofcytokine receptor modulators include, but are not limited to, cytokines,fragments of cytokines, fusion proteins and antibodies thatimmunospecifically binds to a cytokine receptor or a fragment thereof.Further, examples of cytokine receptor modulators include, but are notlimited to, peptides, polypeptides (e.g., soluble cytokine receptors),fusion proteins and antibodies that immunospecifically binds to acytokine or a fragment thereof.

As used herein, the terms “SYNAGIS® fragment”, “antigen-bindingfragment” and like terms used in the context of SYNAGIS® refer to afragment of SYNAGIS® that immunospecifically binds to a RSV antigen.Fragments of SYNAGIS® may be generated by any technique known to thoseskilled in the art. For example, Fab and F(ab′)₂ fragments may beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). F(ab′)₂ fragments contain the complete light chain,and the variable region, the CH1 region and the hinge region of theheavy chain. Preferably, the fragment also binds to a RSV antigen, morepreferably to the same epitope as SYNAGIS®.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent), which is sufficientto reduce the severity, and/or duration of a RSV infection, ameliorateone or more symptoms thereof, prevent the advancement of a RSVinfection, or cause regression of a RSV infection, or which issufficient to result in the prevention of the development, recurrence,onset, or progression of a RSV infection or one or more symptomsthereof, or enhance or improve the prophylactic and/or therapeuticeffect(s) of another therapy (e.g., another therapeutic agent). In aspecific embodiment, an effective amount of a therapeutic or aprophylactic agent reduces one or more of the following steps of a RSVlife cycle: the docking of the virus particle to a cell, theintroduction of viral genetic information into a cell, the expression ofviral proteins, the production of new virus particles and the release ofvirus particles from a cell by at least 5%, preferably at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%. In another specificembodiment, an effective amount of a therapeutic or a prophylactic agentreduces the replication, multiplication or spread of a virus by at least5%, preferably at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

As used herein, the term “epitope” refers to a portion of a RSVpolypeptide or protein having antigenic or immunogenic activity in ananimal, preferably a mammal, and most preferably in a human. An epitopehaving immunogenic activity is a portion of a RSV polypeptide or proteinthat elicits an antibody response in an animal. An epitope havingantigenic activity is a portion of a RSV polypeptide or protein to whichan antibody immunospecifically binds as determined by any method wellknown in the art, for example, by the immunoassays described herein.Antigenic epitopes need not necessarily be immunogenic. Specifically,the epitope of SYNAGIS® is the A antigenic site of the F protein of RSV.

As used herein, the term “excipients” refers to inert substances whichare commonly used as a diluent, vehicle, preservatives, binders, orstabilizing agent for drugs and includes, but not limited to, proteins(e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamicacid, lysine, arginine, glycine, histidine, etc.), fatty acids andphospholipids (e.g., alkyl sulfonates, caprylate, etc.), sufactants(e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g.,sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol,sorbitol, etc.). Also see Remington's Pharmaceutical Sciences (by JosephP. Remington, 18th ed., Mack Publishing Co., Easton, Pa.) which ishereby incorporated in its entirety. Preferably, the excipients impart abeneficial physical property to the formulation, such as increasedprotein stability, increased protein solubility and decreased viscosity.

The term “fragment” as used herein refers to a peptide, polypeptide, orprotein (including an antibody) comprising an amino acid sequence of atleast 5 contiguous amino acid residues, at least 10 contiguous aminoacid residues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least contiguous 80 aminoacid residues, at least contiguous 90 amino acid residues, at leastcontiguous 100 amino acid residues, at least contiguous 125 amino acidresidues, at least 150 contiguous amino acid residues, at leastcontiguous 175 amino acid residues, at least contiguous 200 amino acidresidues, or at least contiguous 250 in amino acid residues of the aminoacid sequence of another polypeptide or protein. In a specificembodiment, a fragment of a protein or polypeptide retains at least onefunction of the protein or polypeptide. In another embodiment, afragment of a protein or polypeptide retains at least two, three or fourfunctions of the protein or polypeptide. Preferably a fragment of anantibody that immunospecifically binds to a RSV antigen retains theability to bind to a RSV antigen.

As used herein, the term “fusion protein” refers to a polypeptide orprotein that comprises an amino acid sequence of a first protein,polypeptide or fragment, analog or derivative thereof, and an amino acidsequence of a heterologous protein or polypeptide (i.e., a secondprotein, polypeptide or fragment, analog or derivative thereof differentthan the first protein or functional fragment, analog or derivativethereof). In one embodiment, a fusion protein comprises a prophylacticor therapeutic agent fused to a heterologous protein, polypeptide orpeptide. In accordance with this embodiment, the heterologous protein,polypeptide or peptide may or may not be a different type ofprophylactic or therapeutic agent.

As used herein, the term “human infant” refers to a human less than 24months, preferably less than 16 months, less than 12 months, less than 6months, less than 3 months, less than 2 months, or less than 1 month ofage.

As used herein, the term “human infant born prematurely” refers to ahuman born at less than 40 weeks gestational age, preferably less than35 weeks gestational age, who is less than 6 months old, preferably lessthan 3 months old, more preferably less than 2 months old, and mostpreferably less than 1 month old.

As used herein, the term “high concentration” refers to a concentrationof 50 mg/ml or higher, preferably 95 mg/ml or higher of an antibody orfragment thereof that immunospecifically binds to a RSV antigen, in anantibody formulation.

As used herein, the term “host cell” includes a subject cell transfectedor transformed with a nucleic acid molecule and the progeny or potentialprogeny of such a cell. Progeny of such a cell may not be identical tothe parent cell transfected with the nucleic acid molecule due tomutations or environmental influences that may occur in succeedinggenerations or integration of the nucleic acid molecule into the hostcell genome.

As used herein, the term “immunospecifically binds a RSV antigen” andanalogous terms refer to antibodies or fragments thereof thatspecifically bind to a RSV antigen thereof and do not specifically bindto other polypeptides. Antibodies or fragments that immunospecificallybind a RSV antigen may be cross-reactive with related antigens.Preferably, antibodies or fragments that immunospecifically bind a RSVantigen do not cross-react with other antigens. Antibodies or fragmentsthat immunospecifically bind to and RSV antigen can be identified, forexample, by immunoassays, BIAcore, isothermal titration calorimetry, orother techniques known to those of skilled in the art. An antibody or anantigen-binding fragment thereof binds specifically to a RSV antigenwhen it binds with higher affinity than to any cross-reactive antigen asdetermined using experimental techniques, such as radioimmunoassays(RIA) and enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul,ed., 1989, Fundamental Immunology Second Edition, Raven Press, New Yorkat pages 332–336 for a discussion regarding antibody specificity.

The term “in combination” as used herein refers to the use of more thanone therapies (e.g., prophylactic and/or therapeutic agents). The use ofthe term “in combination” does not restrict the order in which therapies(e.g., prophylactic and/or therapeutic agents) are administered to asubject with a RSV infection. A first therapy (e.g., a prophylactic ortherapeutic agent) can be administered prior to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks after) the administration of a secondtherapy (e.g., a prophylactic or therapeutic agent) to a subject with aRSV infection.

As used herein, the term “inorganic salt” refers to any compounds,containing no carbon, that result from replacement of part or all of theacid hydrogen or an acid by a metal or a group acting like a metal andare often used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄, etc.

An “isolated” or “purified” antibody is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of anantibody in which the polypeptide/protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, an antibody that is substantially free of cellularmaterial includes preparations of the antibody having less than about30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of contaminatingprotein. When the antibody is recombinantly produced, it is alsopreferably substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, 10%, or 5% of the volume of the proteinpreparation. When antibody is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, 5% (bydry weight) of chemical precursors or compounds other than antibody ofinterest. In a preferred embodiment of the present invention, antibodiesare isolated or purified.

As used herein, the phrase “low to undetectable levels of aggregation”refers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1%, and most preferably no morethan 0.5%, aggregation by weight protein as measured by high performancesize exclusion chromatography (HPSEC).

As used herein, the term “low to undetectable levels of fragmentation”refers to samples containing equal to or more than 80%, 85%, 90%, 95%,98%, or 99%, of the total protein, for example, in a single peak asdetermined by high performance size exclusion chromatography (HPSEC), orin two (2) peaks (heavy- and light-chains) by reduced Capillary GelElectrophoresis (rCGE), representing the non-degraded SYNAGIS® or anon-degraded fragment thereof which immunospecifically binds to a RSVantigen, and containing no other single peaks having more than 5%, morethan 4%, more than 3%, more than 2%, more than 1%, or more than 0.5% ofthe total protein each. The term “Reduced-Capillary Gel Electrophoresis(CGE)” as used herein refers to capillary gel electrophoresis underreducing conditions sufficient to reduce disulfide bonds in SYNAGIS® oran antigen-binding fragment thereof.

As used herein, the terms “manage”, “managing” and “management” refer tothe beneficial effects that a subject derives from a therapy (e.g.,SYNAGIS® or an antigen-binding fragment thereof), which does not resultin a cure of a RSV infection. In certain embodiments, a subject isadministered one or more therapies (e.g., prophylactic or therapeuticagents) to “manage” a RSV infection or a symptom thereof so as toprevent the progression or worsening of the infection.

As used herein, the term “modified” in the context of modified forms ofSYNAGIS® or an antigen-binding fragment thereof refers to SYNAGIS® or anantigen-binding fragment thereof which has been altered by any methodknown in the art to increase its half life (see, e.g., Section 5.1.1.,infra). SYNAGIS® and antigen-binding fragments thereof with improved invivo half-lives and methods for preparing them are disclosed inInternational Publication No. WO 02/060919, filed Dec. 12, 2001, andU.S. patent application Ser. No. 10/020,354, filed Dec. 12, 2001, bothentitled “Molecules with Extended Half-Lives, Compositions and Uses” andby L. Johnson et al. which are hereby incorporated by reference in theirentireties. The term “modified” in the context of SYNAGIS® or anantigen-binding fragment thereof also refers to SYNAGIS® or anantigen-binding fragment modified by covalent attachment of any type ofmolecule to SYNAGIS® or an antigen-binding fragment thereof. Forexample, but not by way of limitation, SYNAGIS® or an antigen-bindingfragment thereof may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc.

As used herein, the term “pharmaceutically acceptable” means beingapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopia, European Pharmacopia or other generallyrecognized pharmacopia for use in animals, and more particularly inhumans.

As used herein, the term “polyol” refers to a sugar that contains many—OH groups compared to a normal saccharide.

As used herein, the terms “prevent”, “preventing”, and “prevention”refer to the prevention or reduction of the recurrence, onset,development or progression of a RSV infection, or the prevention orreduction of the severity and/or duration of a RSV infection or one ormore symptoms thereof.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a RSVinfection or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” refers to SYNAGIS® or an antigen-bindingfragment thereof. In accordance with these embodiments, the antibody orantibody fragment may be a component of a liquid formulation of theinvention. In certain other embodiments, the term “prophylactic agent”does not refer to SYNAGIS® or an antigen-binding fragment thereof. Inyet other embodiments, the term “prophylactic agent” does not refer toantibodies or fragments thereof other than SYNAGIS® thatimmunospecifically bind to a RSV antigen. Preferably, a prophylacticagent is an agent which is known to be useful to, or has been or iscurrently being used to prevent or impede the onset, development,progression, and/or severity of a RSV infection or a symptom thereof.

As used herein, the term “prophylactically effective amount” refers tothe amount of a liquid formulation of the invention which is sufficientto result in the prevention of the development, recurrence, onset orprogression of a RSV infection. In a specific embodiment, aprophylactically effective amount of a prophylactic agent reduces one ormore of the following steps of a RSV life cycle: the docking of thevirus particle to a cell, the introduction of viral genetic informationinto a cell, the expression of viral proteins, the production of newvirus particles and the release of virus particles from a cell by atleast 5%, preferably at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100%. In another specific embodiment, a prophylactically effectiveamount of a prophylactic agent reduces the replication, multiplicationor spread of a virus by at least 5%, preferably at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100%.

As used herein, the term “RSV antigen” refers to a RSV protein,polypeptide or peptide to which an antibody immunospecifically binds.

As used herein, the term “saccharide” refers to a class of moleculesthat are polyhydric alcohols. Saccharides are commonly referred to ascarbohydrates and may contain different amounts of sugar (saccharide)units, e.g., monosaccharides, dissacharides and polysacchorides.

The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, polynucleotides, polynucleotide analogues, nucleotides,nucleotide analogues, organic or inorganic compounds (i.e., includingheterorganic and/or ganometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

As used herein, The terms “stability” and “stable” in the context of aliquid formulation comprising SYNAGIS® or an antigen-binding fragmentthereof refer to the resistance of SYNAGIS® or an antigen-bindingfragment thereof in the formulation to thermal and chemical unfolding,aggregation, degradation or fragmentation under given manufacture,preparation, transportation and storage conditions. The “stable”formulations of the invention retain biological activity equal to ormore than 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% under givenmanufacture, preparation, transportation and storage conditions. Thestability of SYNAGIS® or an antigen-binding fragment thereof can beassessed by degrees of aggregation, degradation or fragmentation bymethods known to those skilled in the art, including but not limited toreduced Capillary Gel Electrophoresis (rCGE), Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDS-PAGE) and HPSEC, compared to areference, that is, a commercially available lyophilized SYNAGIS®reconstituted to 100 mg/ml in 47 mM histidine/3 mM glycine buffer with5.6% mannitol at pH 6.0. The reference regularly gives a single peak(≧97% area) by HPSEC. The overall stability of a liquid formulationcomprising SYNAGIS® or an antigen-binding fragment thereof thatimmunospecifically binds to a RSV antigen can be assessed by variousimmunological assays including, for example, ELISA and radioimmunoassayusing the specific epitope of RSV.

As used herein, the term “SYNAGIS® standard reference” or analogousterms refer to commercially available lyophilized SYNAGIS®, as describedin the Physicians' Desk Reference, 56^(th) edition, 2002. ReconstitutedSYNAGIS® may contain, e.g., the following excipients: 47 mM histidine,3.0 mM glycine and 5.6% manitol and the active ingredient, the antibody,at a concentration of 100 milligrams per ml solution.

The terms “subject” and “patient” are used interchangeably herein. Asused herein, the terms “subject” and “subjects” refer to an animal,preferably a mammal including a non-primate (e.g., a cow, pig, horse,cat, dog, rat, and mouse) and a primate (e.g., a chimpanzee, a monkeysuch as a cynomolgous monkey, and a human), and more preferably a human.

As used herein, the term “substantially free of surfactant” refers to aformulation of SYNAGIS® or an antigen-binding fragment thereof, saidformulation containing less than 0.0005%, less than 0.0003%, or lessthan 0.0001% of surfactants and/or less than 0.0005%, less than 0.0003%,or less than 0.0001% of surfactant.

As used herein, the term “substantially free of inorganic salts” refersto a formulation of SYNAGIS® or an antigen-binding fragment thereof,said formulation containing less than 0.0005%, less than 0.0003%, orless than 0.0001% of inorganic salts.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures; namely, they are composed of groups ofopposing solubility tendencies, typically an oil-soluble hydrocarbonchain and a water-soluble ionic group. Surfactants can be classified,depending on the charge of the surface-active moiety, into anionic,cationic, and nonionic surfactants. Surfactants are often used aswetting, emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment, management oramelioration a RSV infection or one or more symptoms thereof. In certainembodiments, the term “therapeutic agent” refers to SYNAGIS® or anantigen-binding fragment thereof. In accordance with these embodiments,the antibody or antibody fragment may be a component of a liquidformulation of the invention. In certain other embodiments, the term“therapeutic agent” does not refer to SYNAGIS® or an antigen-bindingfragment thereof. In yet other embodiments, the term “therapeutic agent”does not refer to antibodies or fragments thereof, other than SYNAGIS®that immunospecifically bind to a RSV antigen. Preferably, a therapeuticagent is an agent which is known to be useful for, or has been or iscurrently being used for the treatment, management or amelioration of aRSV infection or one or more symptoms thereof.

As used herein, the term “therapeutically effective amount” refers tothe amount of a liquid formulation of the invention that reduces orameliorates the progression, severity, and/or duration of a RSVinfection, and/or ameliorates one or more symptoms associated with a RSVinfection. With respect to the treatment of a RSV infection, atherapeutically effective amount refers to the amount of a therapeuticagent sufficient to reduce or inhibit the replication of a virus,inhibit or reduce the infection of cell with the virus, inhibit orreduce the production of the viral particles, inhibit or reduce therelease of viral particles, inhibit or reduce the spread of the virus toother tissues or subjects, or ameliorate one or more symptoms associatedwith the infection. In a specific embodiment, a therapeuticallyeffective amount of a therapeutic agent reduces one or more of thefollowing steps of a RSV life cycle: the docking of the virus particleto a cell, the introduction of viral genetic information into a cell,the expression of viral proteins, the production of new virus particlesand the release of virus particles from a cell by at least 5%,preferably at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%.In another specific embodiment, a therapeutically effective amount of atherapeutic agent reduces the replication, multiplication or spread of avirus by at least 5%, preferably at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 100%.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s) and/or agent(s) that can be used in theprevention, treatment, management or amelioration of a RSV infection orone or more symptoms thereof. In certain embodiments, the terms“therapy” and “therapies” refer to biological therapy, and/or othertherapies useful for the treatment of a RSV infection known to medicalpersonnel skilled.

As used herein, the terms “treat”, “treating” and “treatment” refer tothe reduction or amelioration of the progression, severity, and/orduration of a RSV infection and/or reduces or ameliorates one or moresymptoms of a RSV infection. In specific embodiments, such terms referto the reduction or inhibition of the replication of a respiratorysyncytial virus (RSV), the inhibition or reduction in the spread of arespiratory syncytial virus (RSV) to other tissues or subjects, theinhibition or reduction of infection of a cell with a respiratorysyncytial virus (RSV), or the amelioration of one or more symptomsassociated with a respiratory syncytial virus (RSV) infection.

A used herein, a “protocol” includes dosing schedules and dosingregimens. The protocols herein are methods of use and includeprophylactic and therapeutic protocols.

As used herein, the term “T cell receptor modulator” refers to an agentwhich modulates the phosphorylation of a T cell receptor, the activationof a signal transduction pathway associated with a T cell receptorand/or the expression of a particular protein such as a cytokine. Suchan agent may directly or indirectly modulate the phosphorylation of a Tcell receptor, the activation of a signal transduction pathwayassociated with a T cell receptor, and/or the expression of a particularprotein such as a cytokine. Examples of T cell receptor modulatorsinclude, but are not limited to, peptides, polypeptides, proteins,fusion proteins and antibodies which immunospecifically bind to a T cellreceptor or a fragment thereof. Further, examples of T cell receptormodulators include, but are not limited to, proteins, peptides,polypeptides (e.g., soluble T cell receptors), fusion proteins andantibodies that immunospecifically binds to a ligand for a T cellreceptor or a fragment thereof.

As used herein, the term “very little to no loss of the biologicalactivities” refers to antibody activities, including specific bindingabilities of SYNAGIS® or an antigen-binding fragment as measured byvarious immunological assays, including, but not limited to ELISAs andradioimmunoassays. In one embodiment, SYNAGIS® or an antigen-bindingfragment of the liquid formulations of the invention retainapproximately 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 98% of the ability to immunospecifically bind to a RSV antigen ascompared to a reference antibody or antibody fragment as measured by animmunological assay known to one of skill in the art or describedherein. For example, an ELISA based assay may be used to compare theability of a liquid formulation of SYNAGIS® or an antigen-bindingfragment thereof to immunospecifically bind to a RSV antigen to aSYNAGIS® reference standard. In this assay, plates are coated with a RSVantigen and the binding signal of a set concentration of a SYNAGIS®reference standard is compared to the binding signal of the sameconcentration of a test antibody or antibody fragment.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the outline for preparing purifiedSYNAGIS®.

FIG. 2 shows the clinical study flow chart for comparing the liquidformulation of SYNAGIS® with the lyophilized formulation of SYNAGIS®.

5. DETAILED DESCRIPTION OF THE INVENTION

The liquid formulations of the present invention provide a ready-to-usepreparation of SYNAGIS® or an antigen-binding fragment thereof foradministering to a subject without having to reconstitute thepreparation accurately and aseptically and waiting for a period of timeuntil the solution clarifies before administering the formulation to thesubject. It simplifies the procedure of administering the formulation toa subject for a healthcare professional. Furthermore, due to its highstability during the storage, the formulations of the present inventioncan contain SYNAGIS® or an antigen-binding fragment thereof atconcentrations in the range of about 15 mg/ml to about 300 mg/ml withoutcausing an adverse effect on the biological activity(ies) of SYNAGIS® oran antigen-binding fragment thereof due to protein aggregation and/orfragmentation during a prolonged storage. Such stability not onlyensures the efficacy of SYNAGIS® or an antigen-binding fragment thereofbut also reduces possible risks of causing adverse effects on a subject.In addition, the manufacturing process of the liquid formulations of thepresent invention is simplified and more efficient than themanufacturing process for the lyophilized version because all stages ofthe manufacturing of the liquid formulations are carried out in anaqueous solution, involving no drying process, such as lyophilizationand freeze-drying. Accordingly, it is more cost effective as well.

5.1 SYNAGIS® Liquid Formulations

The liquid formulations of the present invention provide antibodyformulations which are substantially free of surfactant, inorganicsalts, and/or other excipients and yet exhibit high stability duringlong periods of storage. In a specific embodiment, such antibodyformulations are homogeneous. The formulations of the present inventioncomprise histidine at concentrations between 1 and 100 mM and SYNAGIS®or an antigen-binding fragment thereof at concentrations of about 15mg/ml to about 300 mg/ml. In one embodiment, the formulations of theinvention do not comprise other ingredients except for water or suitablesolvents. In another specific embodiment, a modified form of SYNAGIS®antibody or an antigen-binding fragment thereof having improvedhalf-life and/or affinity is used in the liquid formulations of theinvention.

The concentration of SYNAGIS® or an antigen-binding fragment thereofwhich is included in the liquid formulations of the invention, is atleast 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml,at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least 50mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, atleast 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, at least 105mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml, atleast 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, at least 140mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 250 mg/ml, or atleast 300 mg/ml.

The concentration of histidine which is included in the liquidformulations of the invention ranges from about 1 mM to about 100 mM,about 10 mM to about 50 mM, about 20 mM to about 30 mM, or about 23 mMto about 27 mM, and is most preferably about 25 mM. Histidine can be inthe form of L-histidine, D-histidine, or a mixture thereof, butL-histidine is the most preferable. Histidine can be also in the form ofhydrates. Histidine may be used in a form of pharmaceutically acceptablesalt, such as hydrochloride (e.g., monohydrochloride anddihydrochloride), hydrobromide, sulfate, acetate, etc. The purity ofhistidine should be at least 98%, preferably at least 99%, and mostpreferably at least 99.5%.

The pH of the formulation should not be equal to the isoelectric pointof the particular antibody to be used in the formulation (e.g., theisoelectric point of SYNAGIS® ranges from 8.65 to 9.43) and may rangefrom about 5.0 to about 7, preferably about 5.5 to about 6.5, morepreferably about 5.8 to about 6.2, and most preferably about 6.0.

In addition to histidine and SYNAGIS® or an antigen-binding fragmentthereof, the formulations of the present invention may further compriseglycine at a concentration of less than 150 mM, less than 100 mM, lessthan 50 mM, less than 3.0 mM, less than 2.0 mM, or less than 1.8 mM, andmost preferably 1.6 mM. The amount of glycine in the formulation shouldnot cause a significant buffering effect so that antibody precipitationat its isoelectric point can be avoided. Glycine may be also used in aform of a pharmaceutically acceptable salt, such as hydrochloride,hydrobromide, sulfate, acetate, etc. The purity of glycine should be atleast 98%, preferably at least 99%, and most preferably 99.5%. In aspecific embodiment, glycine is not included in the liquid formulationsof the present invention.

Optionally, the formulations of the present invention may furthercomprise other excipients, such as saccharides (e.g., sucrose, mannose,trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). In oneembodiment, the other excipient is a saccharide. In a specificembodiment, the saccharide is sucrose, which is at a concentration rangebetween about 1% and about 20%, preferably about 5% and about 15%, morepreferably about 8% and 10%. In another embodiment, the other excipientis a polyol. Preferably, however, the liquid formulations of the presentinvention do not contain mannitol. In a specific embodiment, the polyolis polysorbate (e.g., Tween 20), which is at a concentration rangebetween about 0.001% and about 1%, preferably, about 0.01 to about 0.1.

The liquid formulations of the present invention exhibit stability atthe temperature ranges of 38° C.–42° C. for at least 60 days and, insome embodiments, not more than 120 days, of 20° C.–24° C. for at least1 year, of 2° C.–8° C. (in particular, at 4° C.) for at least 3 years,at least 4 years, or at least 5 years and at −20° C. for at least 3years, at least 4 years, or at least 5 years, as assessed by highperformance size exclusion chromatography (HPSEC). Namely, the liquidformulations of the present invention have low to undetectable levels ofaggregation and/or fragmentation, as defined herein, after the storagefor the defined periods as set forth above. Preferably, no more than 5%,no more than 4%, no more than 3%, no more than 2%, no more than 1%, andmost preferably no more than 0.5%, of SYNAGIS® or an antigen-bindingfragment thereof forms an aggregate as measured by HPSEC, after thestorage for the defined periods as set forth above. Furthermore, liquidformulations of the present invention exhibit almost no loss inbiological activity(ies) of SYNAGIS® or an antigen-binding fragmentthereof during the prolonged storage under the condition describedabove, as assessed by various immunological assays including, forexample, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassayto measure the RSV antigen-binding ability of SYNAGIS® or anantigen-binding fragment thereof, or, for example, by a C3a/C4a assay tomeasure the complement activating ability of SYNAGIS® or anantigen-binding fragment thereof. The liquid formulations of the presentinvention retain after the storage for the above-defined periods morethan 80%, more than 85%, more than 90%, more than 95%, more than 98%,more than 99%, or more than 99.5% of the initial biologicalactivity(ies) prior to the storage.

The liquid formulations of the present invention can be prepared as unitdosage forms. For example, a unit dosage per vial may contain 1 ml, 2ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml ofdifferent concentrations of SYNAGIS® or an antigen-binding fragmentthereof ranging from about 15 mg/ml to about 300 mg/ml concentration ofSYNAGIS® or an antigen-binding fragment thereof which immunospecificallybinds to a RSV. If necessary, these preparations can be adjusted to adesired concentration by adding a sterile diluent to each vial.

5.1.1 SYNAGIS®

The invention relates to liquid formulations comprising SYNAGIS® or anantigen-binding fragment thereof. In a preferred embodiments, theinvention provides liquid formulations of SYNAGIS®, a humanizedmonoclonal antibody which neutralizes a broad range of RSV isolates. Theamino acid sequence of SYNAGIS® is disclosed, e.g., in Johnson et al.,1997, J. Infectious Disease 176:1215–1224, and U.S. Pat. No. 5,824,307,and its V_(H)CDRs and V_(L)CDRs are shown in Table 1, infra. Theproperties and uses of SYNAGIS® are also disclosed in, e.g., otherapplications, see, e.g., U.S. patent application Ser. No. 09/724,396filed Nov. 28, 2000; U.S. patent application Ser. No. 09/996,265 filedNov. 28, 2001 and U.S. patent application Ser. No. 10/403,180 filed Mar.31, 2003, all of which are incorporated herein by reference.

TABLE 1 CDR Sequences of SYNAGIS ® CDR Sequence SEQ ID NO: VH1 TSGMSVG 1VH2 DIWWDDKKDYNPSLKS 2 VH3 SMITNWYFDV 3 VL1 KCQLSVGYMH 4 VL2 DTSKLAS 5VL3 FQGSGYPFT 6

In addition, the present invention also encompasses stable liquidformulations of modified forms of SYNAGIS® or an antigen-bindingfragment thereof that have improved half-lives. In particular, thepresent invention encompasses a modified form of SYNAGIS® or anantigen-binding fragment thereof which has a half-life in a subject,preferably a human, of greater than 3 days, greater than 7 days, greaterthan 10 days, preferably greater than 15 days, greater than 25 days,greater than 30 days, greater than 35 days, greater than 40 days,greater than 45 days, greater than 2 months, greater than 3 months,greater than 4 months, or greater than 5 months. By prolonging thehalf-lives of SYNAGIS® and antigen-binding fragments thereof, it ispossible to reduce the amount and/or frequency of dosing of the antibodyor antigen-binding fragment.

To prolong the serum circulation of an antibody in vivo, varioustechniques can be used. For example, inert polymer molecules, such ashigh molecular weight polyethyleneglycol (PEG), can be attached to anantibody with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N- or C-terminus of theantibody or via epsilon-amino groups present on lysine residues. Linearor branched polymer derivatization that results in minimal loss ofbiological activity can be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methods knownto those of skilled in the art, for example, by immunoassays describedherein.

An antibody having an increased half-life in vivo can also be generatedby introducing one or more amino acid modifications (i.e.,substitutions, insertions or deletions) into an IgG constant domain, orFcRn binding fragment thereof (preferably a Fe or hinge Fe domainfragment). See, e.g., International Publication No. WO 98/23289;International Publication No. WO 97/34631; and U.S. Pat. No. 6,277,375,each of which is incorporated herein by reference in its entirety.SYNAGIS® and antigen-binding fragments thereof with improved in vivohalf-lives and methods for preparing them are disclosed in InternationalApplication WO 02/060919, filed Dec. 12, 2001, and U.S. patentapplication Ser. No. 10/020,354, filed Dec. 12, 2001, both entitled“Molecules with Extended Half-Lives, Compositions and Uses” and by L.Johnson et al which are hereby incorporated by reference in theirentireties.

Further, an antibody can be conjugated to albumin in order to make theantibody or an antigen-binding fragment thereof more stable in vivo orhave a longer half life in vivo. The techniques are well known in theart, see e.g., International Publication Nos. WO 93/15199, WO 93/15200,and WO 01/77137; and European Patent No. EP 413, 622, all of which areincorporated herein by reference.

The invention further comprises liquid formulations of SYNAGIS® orantigen-binding fragments thereof that have been modified, for example,by glycosylation, acetylation, pegylatin, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc., andretain RSV antigen-binding activity.

5.1.2 Antibody Conjugates

The present invention encompasses the use of liquid formulations ofSYNAGIS® or an antigen-binding fragment thereof (including modifiedforms that have increased in vivo half-lives) that conjugated to one ormore moieties, including but not limited to, peptides, polypeptides,proteins, fusion proteins, nucleic acid molecules, small molecules,mimetic agents, synthetic drugs, inorganic molecules, and organicmolecules.

The present invention encompasses the use of liquid formulations ofSYNAGIS® recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous protein orpolypeptide (or antigen-binding fragment, preferably to a polypeptide ofat least 10, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90 or at least 100 aminoacids) to generate fusion proteins. The fusion does not necessarily needto be direct, but may occur through linker sequences. For example, anantibody may be used to target a heterologous polypeptide to aparticular cell type, either in vitro or in vivo, by fusing orconjugating the antibody to another antibody specific for particularcell surface receptors. An antibody fused or conjugated to aheterologous polypeptide may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g.,International publication No. WO 93/21232; European Patent No. EP439,095; Naramura et al., 1994, Immunol. Lett. 39:91–99; U.S. Pat. No.5,474,981; Gillies et al., 1992, PNAS 89:1428–1432; and Fell et al.,1991, J. Immunol. 146:2446–2452, which are incorporated by reference intheir entireties.

The present invention further includes compositions comprising aheterologous protein, peptide or polypeptide fused or conjugated to anantigen-binding fragment of SYNAGIS®. For example, a heterologouspolypeptides may be fused or conjugated to a Fab fragment, Fd fragment,Fv fragment, or F(ab)₂ fragment. Methods for fusing or conjugating apolypeptide to antibody portion are known in the art. See, e.g., U.S.Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and5,112,946; European Patent Nos. EP 307,434 and EP 367,166; Internationalpublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,Proc. Natl. Acad. Sci. USA 88: 10535–10539; Zheng et al., 1995, J.Immunol. 154:5590–5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA89:11337–11341 (all references are incorporated herein by reference intheir entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of SYNAGIS® or fragments thereof (e.g.,an antibody or an antigen-binding fragment thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724–33; Harayama, 1998, TrendsBiotechnol. 16(2):76–82; Hansson et al., 1999, J. Mol. Biol. 287:265–76;and Lorenzo and Blasco, 1998, Biotechniques 24(2):308–313 (each of thesepatents and publications are hereby incorporated by reference in itsentirety). SYNAGIS® or an antigen-binding fragment thereof, or thenucleic acid encoding SYNAGIS® or an antigen-binding fragment thereof,may be altered by being subjected to random mutagenesis by error-pronePCR, random nucleotide insertion or other methods prior torecombination. SYNAGIS® or an antigen-binding fragment thereof may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

Moreover, SYNAGIS® or an antigen-binding fragment thereof can be fusedto a marker sequence, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., 1989, Proc. Natl. Acad. Sci. USA 86:821–824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell 37:767) and the “flag” tag.

The present invention also encompasses the liquid formulations ofSYNAGIS® or an antigen-binding fragment thereof conjugated to adiagnostic or detectable agent or any other molecule for which serumhalf-life is desired to be increased. Such an antibody can be useful formonitoring or prognosing the development or progression of a RSVinfection as part of a clinical testing procedure, such as determiningthe efficacy of a particular therapy. Such diagnosis and detection canbe accomplished by coupling SYNAGIS® or an antigen-binding fragmentthereof to a detectable substance including, but not limited to, variousenzymes, such as but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹ Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes. Thedetectable substance may be coupled or conjugated either directly toSYNAGIS® or an antigen-binding fragment thereof or indirectly, throughan intermediate (such as, for example, a linker known in the art) usingtechniques known in the art. See, e.g., U.S. Pat. No. 4,741,900 formetal ions which can be conjugated to antibodies for use as adiagnostics according to the present invention.

The present invention further encompasses uses of SYNAGIS® or anantigen-binding fragment thereof conjugated to a therapeutic moiety. Anantibody or antigen-binding fragment may be conjugated to a therapeuticmoiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxel, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticmoieties include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, camustine (BCNU) and lomustine (CCNU)),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin));anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); Auristatin molecules(e.g., auristatin PHE, bryostatin 1, solastatin 10, see Woyke et al.,Antimicrob. Agents Chemother. 46:3802–8 (2002), Woyke et al.,Antimicrob. Agents Chemother. 45:3580–4 (2001), Mohammad et al.,Anticancer Drugs 12:735–40 (2001), Wall et al., Biochem. Biophys. Res.Commun. 266:76–80 (1999), Mohammad et al., Int. J. Oncol. 15:367–72(1999), all of which are incorporated herein by reference); anti-mitoticagents (e.g., vincristine and vinblastine); hormones (e.g.,glucocorticoids, progestatins, androgens, and estrogens); DNA repairenzyme inhibitors (e.g., etoposide or topotecan); kinase inhibitors(e.g., compound ST1571, imatinib mesylate (Kantarjian et al., ClinCancer Res. 8(7):2167 76 (2002)), and those compounds disclosed in U.S.Pat. Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242,6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877,5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904,5,840,745, 5,728,868, 5,648,239, and 5,587,459); farnesyl transferaseinhibitors (e.g., R115777, BMS 214662, and those disclosed by, forexample, U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960,6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581,6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765,6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140,6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193,6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366,6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870,6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582,6,051,574, and 6,040,305); topoisomerase inhibitors (e.g., camptothecin,irinotecan, SN 38, topotecan, 9 aminocamptothecin, GG 211 (GI 147211),DX 8951f; IST 622, rubitecan, pyrazoloacridine, XR 5000, saintopin,UCE6, UCE1022, TAN 1518A, TAN 1518B, KT6006, KT6528, ED 110, NB 506, ED110, NB 506, rebeccamycin, and bulgarein); DNA minor groove binders suchas Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronine;epiberberine; coralyne; beta lapachone; BC 41; and pharmaceuticallyacceptable salts, solvates, clathrates, and prodrugs thereof (See, e.g.,Rothenberg, M. L., Annals of Oncology 8:837 855(1997); and Moreau etal., J. Med. Chem. 41:1631 1640(1998)). Therapeutic moieties may also beantisense oligonucleotides (e.g., those disclosed in the U.S. Pat. Nos.6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709);immunomodulators (e.g., antibodies and cytokines); antibodies (e.g.,rituximab (Rituxan®), calicheamycin (Mylotarg®), ibritumomab tiuxetan(Zevalin®), and tositumomab (Bexxar®)); and adnosine deaminaseinhibitors (e.g., Fludarabine phosphate and 2 Chlorodeoxyadenosine).

Further, an antibody or an antigen-binding fragment thereof may beconjugated to a therapeutic moiety or drug moiety that modifies a givenbiological response. Therapeutic moiety or drug moieties are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, TNF-β,AIM I (see, International publication No. WO 97/33899), AIM II (see,International Publication No. WO 97/34911), Fas Ligand (Takahashi etal., 1994, J. Immunol., 6:1567–1574), and VEGF (see, Internationalpublication No. WO 99/23105); or, a biological response modifier suchas, for example, a lymphokine (e.g., interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-4 (“IL-4”), interleukin-6 (“IL-6”),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12),interferon-α, β, γ, granulocyte macrophage colony stimulating factor(“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or agrowth factor (e.g., growth hormone (“GH”)).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, e.g., alpha-emitters such as ²¹³Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹³¹In, ¹³¹LU, ¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483–90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553–7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943–50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623–53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475–506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303–16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119–58.

Alternatively, SYNAGIS® or an antigen-binding fragment thereof can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980, which is incorporatedherein by reference in its entirety.

SYNAGIS® or an antigen-binding fragment thereof may also be attached tosolid supports, which are particularly useful for immunoassays orpurification of the target antigen. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

The therapeutic moiety or drug conjugated to SYNAGIS® or anantigen-binding fragment thereof should be chosen to achieve the desiredprophylactic or therapeutic effect(s) for a RSV infection in a subject.A clinician or other medical professional should consider the followingwhen deciding on which therapeutic moiety or drug to conjugate toSYNAGIS® or an antigen-binding fragment thereof: the severity of theinfection, and the condition of the subject.

SYNAGIS® or an antigen-binding fragment thereof, with or without atherapeutic moiety conjugated to it, can be used as a therapeutic.

5.2 Method of Preparing the Antibody Formulations

The present invention provides methods for preparing liquid formulationsof SYNAGIS® or an antigen-binding fragment thereof. FIG. 1 is aschematic diagram showing the outline for preparing purified SYNAGIS®.The methods for preparing liquid formulations of the present inventioncomprise: purifying the antibody from conditioned medium (either singlelots or pooled lots of medium) and concentrating a fraction containingthe purified SYNAGIS® to a final antibody concentration of from about 15mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml,about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about100 mg/ml, about 150 mg/ml, about 200 mg/ml, about 250 mg/ml, or about300 mg/ml using a semipermeable membrane with an appropriate molecularweight (MW) cutoff (e.g., 30 kD cutoff for whole antibody molecules andF(ab′)₂ fragments; and 10 kD cutoff for antibody fragments, such as Fabfragments) and diafiltrating the concentrated antibody fraction into theformulation buffer using the same membrane. The formulation buffer ofthe present invention comprises histidine at a concentration from about1 mM to about 100 mM, about 10 mM to about 50 mM, about 20 mM to about30 mM, or about 23 mM to about 27 mM, and is most preferably about 25mM. The formulations may further comprise glycine at a concentration ofless than 100 mM, less than 50 mM, less than 3.0 mM, less than 2.0 mM,or less than 1.8 mM, and most preferably of 1.6 mM. The amount ofglycine in the formulation should not cause a significant buffering inorder to avoid antibody precipitation at its isoelectric point. The pHof the formulation may range from about 5.0 to about 7.0, preferablyabout 5.5 to about 6.5, more preferably about 5.8 to about 6.2, and mostpreferably about 6.0. To obtain an appropriate pH for a particularantibody, it is preferable that histidine (and glycine, if added) isfirst dissolved in water to obtain a buffer solution with higher pH thanthe desired pH and then the pH is brought down to the desired level byadding HCl. This way, the formation of inorganic salts (e.g., formationof NaCl when, for example, histidine hydrochloride is used as histidineand pH is raised to a desired level by adding NaOH) can be avoided.

The liquid formulations of the present invention can be prepared as unitdosage forms by preparing a vial containing an aliquot of the liquidformulation for a one-time use. For example, a unit dosage per vial maycontain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15ml, or 20 ml of different concentrations of SYNAGIS® or anantigen-binding fragment thereof ranging from about 15 mg/ml to about300 mg/ml concentration of SYNAGIS® or an antigen-binding fragmentthereof which immunospecifically binds to a RSV. If necessary, thesepreparations can be adjusted to a desired concentration by adding asterile diluent to each vial.

The liquid formulations of the present invention may be sterilized byvarious sterilization methods, including sterile filtration, radiation,etc. In a most preferred embodiment, the diafiltrated antibodyformulation is filter-sterilized with a presterilized 0.2 or 0.22-micronfilter. Sterilized liquid formulations of the present invention may beadministered to a subject to prevent, treat, manage or ameliorate a RSVinfection or one or more symptoms thereof.

Although the invention is directed to liquid non-lyophilizedformulations, it should be noted for the purpose of equivalents that theformulations of the invention may be lyophilized if desired. Thus, theinvention encompasses lyophilized forms of the formulations of theinvention although such lyophilized formulations are not necessary andthus not preferred.

5.3 Methods of Preparing SYNAGIS®

SYNAGIS® and an antigen-binding fragment thereof contained in the liquidformulations of the present invention can be prepared by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or, preferably, by recombinant expression techniques.

The nucleotide sequence encoding the heavy and light chain variabledomains of SYNAGIS® may be obtained from, for example, co-pendingapplication Ser. No. 09/724,396, filed Nov. 28, 2000 and Ser. No.09/996,265, filed Nov. 28, 2001, both by Young et al., and both of whichare incorporated by reference herein in its entirety. See also, U.S.Pat. No. 5,824,307 by Johnson et al. In certain embodiments, a nucleicacid encoding SYNAGIS® or an antigen-binding fragment thereof may bechemically synthesized or assembled from oligonucleotides as well knownin the art, and then amplified by PCR, cloning or other method known inthe art.

Recombinant expression of an antibody (such as SYNAGIS®) requiresconstruction of an expression vector containing a nucleotide sequencethat encodes the antibody. Once a nucleotide sequence encoding anantibody molecule or a heavy or light chain of an antibody, or anantigen-binding fragment thereof has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art as discussed in theprevious sections. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The nucleotide sequence encoding the heavy-chain variableregion, light-chain variable region, both the heavy-chain andlight-chain variable regions, an epitope-binding fragment of the heavy-and/or light-chain variable region, or one or more complementaritydetermining regions (CDRS) of an antibody may be cloned into such avector for expression. Thus-prepared expression vector can be thenintroduced into appropriate host cells for the expression of theantibody. Accordingly, the invention includes host cells containing apolynucleotide encoding SYNAGIS® or an antigen-binding fragment thereof.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl.Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1–2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223,1977), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48:202, 1992), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:8–17, 1980) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357, 1980 and O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418 (Wu andWu, Biotherapy, 3:87–95, 1991; Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573–596, 1993; Mulligan, Science, 260:926–932, 1993; andMorgan and Anderson, Ann. Rev. Biochem., 62: 191–217, 1993; and May, TIBTECH, 11(5):155–215, 1993); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147, 1984). Methods commonly knownin the art of recombinant DNA technology may be routinely applied toselect the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12and 13, Dracopoli et al. (eds), 1994, Current Protocols in HumanGenetics, John Wiley & Sons, NY; and Colberre-Garapin et al., J. Mol.Biol., 150:1, 1981, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, 1987, The useof vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. Academic Press, NewYork). When a marker in the vector system expressing antibody isamplifiable, increasing in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol., Cell. Biol.,3:257, 1983).

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A purification, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard techniques for the purification of proteins. Further,SYNAGIS® or an antigen-binding fragment thereof may be fused toheterologous protein, polypeptide or peptide sequences described hereinor otherwise known in the art to facilitate purification.

Antigen-binding fragments of SYNAGIS® that immunospecifically bind RSVmay be generated by known techniques. For example, Fab and F(ab′)₂fragments may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thecomplete light chain, and the variable region, the CH1 region and thehinge region of the heavy chain.

5.4 Methods of Monitoring the Stability and Aggregation of AntibodyFormulations

There are various methods available for assessing the stability ofprotein formulations, including antibody formulations, based on thephysical and chemical structures of the proteins as well as on theirbiological activities. For example, to study denaturation of proteins,methods such as charge-transfer absorption, thermal analysis,fluorescence spectroscopy, circular dichroism, NMR, and HPSEC, areavailable. See, for example, Wang et al., 1988, J. of Parenteral Science& Technology 42(supp):S4–S26. The rCGE, and HPSEC are the most commonand simplest methods to assess the formation of protein aggregates,protein degradation and protein fragmentation. Accordingly, thestability of the liquid formulations of the present invention may beassessed by these methods.

For example, the stability of the liquid formulations of the presentinvention may be evaluated by HPSEC or rCGE, wherein the percentage areaof the peaks represents the non-degraded SYNAGIS® or non-degradedantigen-binding fragments of SYNAGIS®. In particular, approximately 250μg of SYNAGIS® or an antigen-binding fragment thereof (approximately 25μl of a liquid formulation comprising 10 mg/ml of SYNAGIS® or anantigen-binding fragment thereof) is injected onto a TOSOH TSKG3000SW_(XL) column (7.8 mm×30 cm) fitted with a TSK SW×1 guard column(6.0 mm×4.0 cm), SYNAGIS® or an antigen-binding fragment thereof iseluted isocratically with 0.1 M disodium phosphate containing 0.1 Msodium sulfate and 0.05% sodium azide, at a flow rate of 0.8 to 1.0ml/min. Eluted protein is detected using UV absorbance at 280 nm.SYNAGIS® reference standard is run in the assay as a control, and theresults are reported as the area percent of the product monomer peakcompared to all other peaks excluding the included volume peak observedapproximately at 12 to 14 minutes. Peaks eluting earlier than themonomer peak are recorded as percent aggregate.

The liquid formulations of the present invention exhibit low toundetectable levels of aggregation as measured by HPSEC or rCGE, thatis, no more than 5%, no more than 4%, no more than 3%, no more than 2%,no more than 1%, and most preferably no more than 0.5% aggregate byweight protein, and low to undetectable levels of fragmentation, thatis, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% orhigher, or 99% or higher, or 99.5% or higher of the total peak area inthe peak(s) representing intact antibodies or fragments thereof. In thecase of SDS-PAGE, the density or the radioactivity of each band stainedor labeled with radioisotope can be measured and the % density or %radioactivity of the band representing non-degraded SYNAGIS® orantigen-binding fragments thereof can be obtained.

The stability of the liquid formulations of the present invention can bealso assessed by any assays which measures the biological activitySYNAGIS® or an antigen-binding fragment thereof in the formulation. Thebiological activities of an antibody include, but not limited to,antigen-binding activity, complement-activation activity, Fc-receptorbinding activity, and so forth. Antigen-binding activity of SYNAGIS® oran antigen-binding fragment thereof can be measured by any method knownto those skilled in the art, including but not limited to ELISA,radioimmunoassay, Western blot, and the like. Complement-activationactivity can be measured by a C3a/C4a assay in the system where SYNAGIS®or an antigen-binding fragment thereof is reacted in the presence of thecomplement components with cells expressing a RSV antigen. Also seeHarlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988) (incorporated by reference herein in itsentirety). An ELISA based assay, e.g., may be used to compare theability of a liquid formulation of SYNAGIS® or an antigen-bindingfragment thereof to immunospecifically bind to a RSV antigen to aSYNAGIS® reference standard. In this assay, plates are coated with RSVantigen (in particular, the A antigenic site of F protein of RSV) andthe binding signal of a set concentration of a SYNAGIS® referencestandard is compared to the binding signal of the same concentration ofthe liquid formulation of SYNAGIS® or an antigen-binding fragmentthereof.

The purity of the liquid antibody formulations of the invention may bemeasured by any method well-known to one of skilled in the art such as,e.g., HPSEC. The sterility of the liquid antibody formulations may beassessed as follows: sterile soybean-casein digest medium and fluidthioglycollate medium are inoculated with a test liquid antibodyformulation by filtering the liquid antibody formulation through asterile filter having a nominal porosity of 0.45 μm. When using theSterisure™ or Steritest™ method, each filter device is asepticallyfilled with approximately 100 ml of sterile soybean-casein digest mediumor fluid thioglycollate medium. When using the conventional method, thechallenged filter is aseptically transferred to 100 ml of sterilesoybean-casein digest medium or fluid thioglycollate medium. The mediaare incubated at appropriate temperatures and observed three times overa 14 day period for evidence of bacterial or fungal growth.

5.5 Prophylactic and Therapeutic Utility of the Antibody Formulations

The present invention is also directed to antibody-based therapies whichinvolve administering to a subject, preferably a mammal, most preferablya human, the liquid antibody formulations of the present invention forpreventing, treating, managing or ameliorating a RSV infection or one ormore symptoms thereof. Prophylactic and therapeutic formulations of theinvention comprise SYNAGIS® or an antigen-binding fragment thereof atconcentrations of from about 15 mg/ml to about 300 mg/ml in a solutioncontaining histidine.

The liquid formulations of the invention may comprise modified SYNAGIS®or antigen-binding fragments thereof that have improved in vivohalf-lives compared to known antibodies that immunospecically binds to aRSV antigen (e.g., unmodified SYNAGIS®).

In one embodiment, the liquid formulations of the present invention areadministered to a mammal, preferably a human, to prevent, treat, manageor ameliorate a RSV infection or one or more symptoms thereof. Inanother embodiment, the liquid formulations of the invention areadministered to a human with cystic fibrosis, bronchopulmonarydysplasia, congenital heart disease, congenital immunodeficiency oracquired immunodeficiency, or to a human who has had a bone marrowtransplant to prevent, treat, mange or ameliorate a RSV infection or oneor more symptoms thereof. In another embodiment, the liquid formulationsof the invention are administered to a human infant, preferably a humaninfant born prematurely or a human infant at risk of hospitalization fora RSV infection to prevent, treat, manage or ameliorate a RSV infectionor one or more symptoms thereof. In another embodiment, the liquidformulations of the invention are administered to an elderly person toprevent, treat, manage or ameliorate a RSV infection or one or moresymptoms thereof. In yet another embodiment, the liquid formulations ofthe invention are administered to a subject in an institution or grouphome (e.g., a nursing home or orphanage).

The liquid formulations of the present invention may be used locally orsystemically in the body of a subject prophylactically ortherapeutically. The formulations of the present invention may also beadvantageously utilized in combination with other therapies useful inthe prevention, treatment, management or amelioration of a RSV infection(e.g., a prophylactic or a therapeutic agent other than SYNAGIS®).Non-limiting examples of prophylactic or therapeutic agents that can beused in combination with the liquid formulations of the presentinvention, see Section 5.6, infra.

When one or more other therapies are used, they can be administeredseparately, in any appropriate form and by any suitable route. A liquidformulation of the invention may be administered to a mammal, preferablya human, concurrently with one or more other therapies (e.g., one ormore other prophylactic or therapeutic agents) useful for theprevention, treatment, management or amelioration of a RSV infection orone or more symptoms thereof. The term “concurrently” is not limited tothe administration of therapies at exactly the same time, but rather itis meant that a liquid formulation of the invention and another therapyare administered to a mammal in a sequence and within a time intervalsuch that SYNAGIS® or an antigen-binding fragment thereof contained inthe liquid formulation can act together with the other therapy toprovide an increased benefit than if they were administered otherwise.For example, a liquid formulation of the invention and one or more otherprophylactic or therapeutic agents useful for prevention, treatment,management or amelioration of a RSV infection may be administered at thesame time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect.

In various embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents) useful for prevention, treatment, management oramelioration of a RSV infection or a symptom thereof are administeredless than 1 hour apart, at about 1 hour apart, at about 1 hour to about2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hoursto about 4 hours apart, at about 4 hours to about 5 hours apart, atabout 5 hours to about 6 hours apart, at about 6 hours to about 7 hoursapart, at about 7 hours to about 8 hours apart, at about 8 hours toabout 9 hours apart, at about 9 hours to about 10 hours apart, at about10 hours to about 11 hours apart, at about 11 hours to about 12 hoursapart, no more than 24 hours apart or no more than 48 hours apart. Inpreferred embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents) useful for prevention, treatment, management oramelioration of a RSV infection or a symptom thereof are administeredwithin the same patient visit. In other embodiments, a liquidformulation of the invention and one or more other therapies (e.g., oneor more other prophylactic or therapeutic agents) useful for prevention,treatment, management or amelioration of a RSV infection or a symptomthereof are administered at about 2 to 4 days apart, at about 4 to 6days apart, at about 1 week part, at about 1 to 2 weeks apart, or morethan 2 weeks apart. In preferred embodiments, a liquid formulation ofthe invention and one or more other prophylactic or therapeutic agentsuseful for prevention, treatment, management or amelioration of a RSVinfection or a symptom thereof are administered in a time frame whereboth agents are still active. One skilled in the art would be able todetermine such a time frame by determining the half-life of theadministered agents.

In certain embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents) useful for prevention, treatment, management oramelioration of a RSV infection or a symptom thereof are cyclicallyadministered to a subject. Cycling therapy involves the administrationof a first therapy for a period of time, followed by the administrationof a second therapy and/or third therapy for a period of time andrepeating this sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improves theefficacy of the treatment.

In certain embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents) useful for prevention, treatment, management oramelioration of a RSV infection or a symptom thereof are administered ina cycle of less than about 3 weeks, about once every two weeks, aboutonce every 10 days or about once every week. One cycle can comprise theadministration of a therapy (e.g., a therapeutic or prophylactic agent)by infusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, human orhumanized antibodies, fragments derivatives, or analogs, areadministered to a human patient for therapy or prophylaxis.

5.6 Agents Useful in Combination with SYNAGIS® Formulations

The present invention provides methods for preventing, managing,treating, or ameliorating a RSV infection or one or more symptomsthereof comprising administering to a subject in need thereof a liquidformulation of the invention alone or in combination with one or moretherapies (e.g., one or more prophylactic or therapeutic agents) otherthan SYNAGIS®. The present invention provides methods for preventing,treating, managing or ameliorating a RSV infection or one or moresymptoms thereof comprising administering to a subject in need thereof aliquid formulation of the invention alone or in combination with one ormore therapies (e.g., one or more prophylactic or therapeutic agents)other than SYNAGIS®. The present invention also provides compositionscomprising a liquid formulation of SYNAGIS® or an antigen-bindingfragment thereof and one or more prophylactic or therapeutic agentsother than SYNAGIS® and methods of preventing, treating, managing orameliorating a RSV infection or one or more symptoms thereof utilizingsaid compositions. Therapeutic or prophylactic agents include, but arenot limited to, small molecules, synthetic drugs, peptides,polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotidesincluding, but not limited to, antisense nucleotide sequences, triplehelices, RNA interference (RNAi), and nucleotide sequences encodingbiologically active proteins, polypeptides or peptides) antibodies,synthetic or natural inorganic molecules, mimetic agents, and syntheticor natural organic molecules.

Any therapy which is known to be useful, or which has been used or iscurrently being used for the prevention, management, treatment, oramelioration of a RSV infection or one or more symptoms thereof can beused in combination with a liquid formulation in accordance with theinvention described herein. See, e.g., Gilman et al, Goodman andGilman's: The Pharmacological Basis of Therapeutics, 10th ed.,McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy,Berkow, M. D. et al (eds.), 17th Ed., Merck Sharp & Dohme ResearchLaboratories, Rahway, N.J., 1999; Cecil Textbook of Medicine, 20th Ed.,Bennett and Plum (eds.), W.B. Saunders, Philadelphia, 1996, forinformation regarding therapies (e.g., prophylactic or therapeuticagents) which have been or are currently being used for preventing,treating, managing, or ameliorating a RSV infection or one or moresymptoms thereof. Examples of such agents include, but are not limitedto, immunomodulatory agents, anti-inflammatory agents (e.g.,adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide,flunisolide, fluticasone, triamcinolone, methlyprednisolone,prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids,non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen,diclofenac, and COX-2 inhibitors), pain relievers, leukotreineantagonists (e.g., montelukast, methyl xanthines, zafirlukast, andzileuton), beta2-agonists (e.g., albuterol, biterol, fenoterol,isoetharie, metaproterenol, pirbuterol, salbutamol, terbutalinformoterol, salmeterol, and salbutamol terbutaline), anticholinergicagents (e.g., ipratropium bromide and oxitropium bromide),sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarialagents (e.g., hydroxychloroquine)), and anti-viral agents.

In specific embodiments, a liquid formulation of the invention is usedin combination with a monoclonal or chimeric antibody, or with alymphokine or hematopoietic growth factor (such as, e.g., IL-2, IL-3,IL-4, IL-7, IL-9, IL-10, IL-12, and interferon α, β, and γ), which, forexample, serves to increase the number or activity of effector cellswhich interact with the antibody. A liquid formulation of the presentinvention may also be advantageously utilized in combination with othermonoclonal or chimeric antibodies, or with lymphokines or hematopoieticgrowth factors (such as, e.g., IL-2, IL-3, IL-4, IL-7, IL-9, IL-10,IL-12, and interferon α, β, and γ), which, for example, serve toincrease the immune response. The liquid formulations of the presentinvention may also be advantageously utilized in combination with one ormore drugs used to treat RSV infection such as, for example anti-viralagents. The liquid formulations of the present invention may be used incombination with one or more of the following drugs: NIH-351 (GeminiTechnologies), recombinant RSV vaccine (MedImmune Vaccines, Inc. U.S.Application Nos. 60/358,934 filed Feb. 21, 2002, Ser. No. 10/373,567filed Feb. 21, 2003, Ser. No. 10/371,099 filed Feb. 21, 2003, Ser. No.10/371,122 filed Feb. 21, 2003, Ser. No. 10/371,264 filed Feb. 21, 2003,60/466,181 filed Apr. 25, 2003 and 60/465,811 filed Apr. 25, 2003, allof which are incorporated herein by reference), RSVf-2 (Intracel),F-50042 (Pierre Fabre), T-786 (Trimeris), VP-36676 (ViroPharma), RFI-641(American Home Products), VP-14637 (ViroPharma), PFP-1 and PFP-2(American Home Products), RSV vaccine (Avant Immunotherapeutics), andF-50077 (Pierre Fabre).

5.6.1 Immunomodulatory Agents

Any immunomodulatory agent well-known to one of skilled in the art maybe used in accordance with the methods of the invention to prevent,treat, manage or ameliorate a RSV infection or one or more symptomsthereof. Immunomodulatory agents can affect one or more or all aspectsof the immune response in a subject. Aspects of the immune responseinclude, but are not limited to, the inflammatory response, thecomplement cascade, leukocyte and lymphocyte differentiation,proliferation, and/or effector function, monocyte and/or basophilcounts, and the cellular communication among cells of the immune system.In certain embodiments of the invention, an immunomodulatory agentmodulates one aspect of the immune response. In other embodiments, animmunomodulatory agent modulates more than one aspect of the immuneresponse. In a preferred embodiment of the invention, the administrationof an immunomodulatory agent to a subject inhibits or reduces one ormore aspects of the subject's immune response capabilities. In analternative embodiment of the invention, the immunomodulatory agentenhances one or more aspects of a subject's immune response. In certainembodiments, an immunomodulatory agent is not an anti-inflammatoryagent. In a specific embodiment, an immunomodulatory agent is an agentother than a chemotherapeutic agent.

Examples of immunomodulatory agents include, but are not limited to,proteinaceous agents such as cytokines, peptide mimetics, and antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab or F(ab)₂ fragments or epitope binding fragments), nucleic acidmolecules (e.g., antisense nucleic acid molecules and triple helices),small molecules, organic compounds, and inorganic compounds. Inparticular, immunomodulatory agents include, but are not limited to,methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators.

Examples of T cell receptor modulators include, but are not limited to,anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g.,cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthocloneand OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion(Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)),anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate),anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies(e.g., MEDI-507 (MedImmune, Inc., International Publication Nos. WO02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))),CTLA4-immunoglobulin, and LFA-3TIP (Biogen, International PublicationNo. WO 93/08656 and U.S. Pat. No. 6,162,432).

Examples of cytokine receptor modulators include, but are not limitedto, soluble cytokine receptors (e.g., the extracellular domain of aTNF-α receptor or an antigen-binding fragment thereof, the extracellulardomain of an IL-1β receptor or an antigen-binding fragment thereof, andthe extracellular domain of an IL-6 receptor or an antigen-bindingfragment thereof), cytokines or fragments thereof (e.g., interleukinIL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-15, IL-23, TNF-α, TNF-β, interferon (IFN)-α, IFN-β, IFN-γ, andGM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptorantibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein DesignLabs)), anti-IL-3 receptor antibodies, anti-IL-4 receptor antibodies,anti-IL-6 receptor antibodies, anti-IL-9 receptor antibodies, anti-IL-10receptor antibodies, anti-IL-12 receptor antibodies, anti-IL-13 receptorantibodies, anti-IL-15 receptor antibodies, and anti-IL-23 receptorantibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies,anti-TNF-α antibodies, anti-IL-1β antibodies, anti-IL-3 antibodies,anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)),anti-IL-9 antibodies, anti-IL-12 antibodies, anti-IL-13 antibodies,anti-IL-15 antibodies, and anti-IL-23 antibodies).

In a specific embodiment, a cytokine receptor modulator is IFN, IL-2,IL-3, IL-4, IL-10, IL-12 or an antigen-binding fragment thereof. Inanother embodiment, a cytokine receptor modulator is an anti-IL-10antibody, anti-IL-6 antibody, anti-IL-9 antibody, anti-IL-12 receptorantibody, or anti-TNF-α antibody. In another embodiment, a cytokinereceptor modulator is the extracellular domain of a TNF-α receptor or anantigen-binding fragment thereof.

An immunomodulatory agent may be selected to interfere with theinteractions between the T helper subsets (TH1 or TH2) and B cells toinhibit neutralizing antibody formation. Antibodies that interfere withor block the interactions necessary for the activation of B cells by TH(T helper) cells, and thus block the production of neutralizingantibodies, are useful as immunomodulatory agents in the methods of theinvention. For example, B cell activation by T cells requires certaininteractions to occur (Durie et al., Immunol. Today, 15(9):406–410(1994)), such as the binding of CD40 ligand on the T helper cell to theCD40 antigen on the B cell, and the binding of the CD28 and/or CTLA4ligands on the T cell to the B7 antigen on the B cell. Without bothinteractions, the B cell cannot be activated to induce production of theneutralizing antibody.

The CD40 ligand (CD40L)-CD40 interaction is a desirable point to blockthe immune response because of its broad activity in both T helper cellactivation and function as well as the absence of redundancy in itssignaling pathway. Thus, in a specific embodiment of the invention, theinteraction of CD40L with CD40 is transiently blocked at the time ofadministration of one or more of the immunomodulatory agents. This canbe accomplished by treating with an agent which blocks the CD40 ligandon the TH cell and interferes with the normal binding of CD40 ligand onthe T helper cell with the CD40 antigen on the B cell. An antibody toCD40 ligand (anti-CD40L) (available from Bristol-Myers Squibb Co; see,e.g., European patent application 555,880, published Aug. 18, 1993) or asoluble CD40 molecule can be selected and used as an immunomodulatoryagent in accordance with the methods of the invention.

An immunomodulatory agent may be selected to inhibit the interactionbetween TH 1 cells and cytotoxic T lymphocytes (“CTLs”) to reduce theoccurrence of CTL-mediated killing. An immunomodulatory agent may beselected to alter (e.g., inhibit or suppress) the proliferation,differentiation, activity and/or function of the CD4⁺ and/or CD8⁺ Tcells. For example, antibodies specific for T cells can be used asimmunomodulatory agents to deplete, or alter the proliferation,differentiation, activity and/or function of CD4⁺ and/or CD8⁺ T cells.

In one embodiment, an immunomodulatory agent which reduces or inhibitsone or more biological activities (e.g., the differentiation,proliferation, and/or effector functions) of TH0, TH1, and/or TH2subsets of CD4⁺ T helper cells is administered to a subject with a RSVinfection in accordance with the methods of the invention. One exampleof such an immunomodulatory agent is IL-4. IL-4 enhancesantigen-specific activity of TH2 cells at the expense of the TH1 cellfunction (see, e.g., Yokota et al, 1986 Proc. Natl. Acad. Sci., USA,83:5894–5898; and U.S. Pat. No. 5,017,691). Other examples ofimmunomodulatory agents that affect the biological activity (e.g.,proliferation, differentiation, and/or effector functions) of T-helpercells (in particular, TH1 and/or TH2 cells) include, but are not limitedto, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-23, andinterferon (IFN)-γ.

In another embodiment, an immunomodulatory agent administered to asubject with a RSV infection in accordance with the methods of theinvention is a cytokine that prevents antigen presentation. In aspecific embodiment, an immunomodulatory agent used in the methods ofthe invention is IL-10. IL-10 also reduces or inhibits macrophage actionwhich involves bacterial elimination.

In accordance with the invention, one or more immunomodulatory agentsare administered to a subject with a RSV infection prior to, subsequentto, or concomitantly with a liquid formulation of SYNAGIS® or anantigen-binding fragment thereof. Preferably, one or moreimmunomodulatory agents are administered in combination with a liquidformulation of SYNAGIS® or an antigen-binding fragment thereof to asubject with a RSV infection to reduce or inhibit one or more aspects ofthe immune response as deemed necessary by one of skilled in the art.Any technique well-known to one skilled in the art can be used tomeasure one or more aspects of the immune response in a particularsubject, and thereby determine when it is necessary to administer animmunomodulatory agent to said subject. In a preferred embodiment, amean absolute lymphocyte count of approximately 500 cells/mm³,preferably 600 cells/mm³, 650 cells/mm³, 700 cells/mm³, 750 cells/mm³,800 cells/mm³, 900 cells/mm³, 1000 cells/mm³, 1100 cells/mm³, or 1200cells/mm³ is maintained in a subject. In another preferred embodiment, asubject with a RSV infection is not administered an immunomodulatoryagent if their absolute lymphocyte count is 500 cells/mm³ or less, 550cells/mm³ or less, 600 cells/mm³ or less, 650 cells/mm³ or less, 700cells/mm³ or less, 750 cells/mm³ or less, or 800 cells/mm³ or less.

In a specific embodiment, one or more immunomodulatory agents areadministered in combination with a liquid formulation of SYNAGIS® or anantigen-binding fragment thereof to a subject with a RSV infection so asto transiently reduce or inhibit one or more aspects of the immuneresponse. Such a transient inhibition or reduction of one or moreaspects of the immune system can last for hours, days, weeks, or months.Preferably, the transient inhibition or reduction in one or more aspectsof the immune response lasts for a few hours (e.g., 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours, 36hours, or 48 hours), a few days (e.g., 3 days, 4 days, 5 days, 6 days, 7days, or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks, 5 weeks or 6weeks). The transient reduction or inhibition of one or more aspects ofthe immune response enhances the prophylactic and/or therapeuticeffect(s) of a liquid formulation of SYNAGIS® or an antigen-bindingfragment thereof.

In a preferred embodiment, proteins, polypeptides or peptides (includingantibodies) that are utilized as immunomodulatory agents are derivedfrom the same species as the recipient of the proteins, polypeptides orpeptides so as to reduce the likelihood of an immune response to thoseproteins, polypeptides or peptides. In another preferred embodiment,when the subject is a human, the proteins, polypeptides, or peptidesthat are utilized as immunomodulatory agents are human or humanized.

Nucleic acid molecules encoding proteins, polypeptides, or peptides withimmunomodulatory activity or proteins, polypeptides, or peptides withimmunomodulatory activity can be administered to a subject with a RSVinfection in accordance with the methods of the invention. Further,nucleic acid molecules encoding derivatives, analogs, or fragments ofproteins, polypeptides, or peptides with immunomodulatory activity, orderivatives, analogs, or fragments of proteins, polypeptides, orpeptides with immunomodulatory activity can be administered to a subjectwith a RSV infection in accordance with the methods of the invention.Preferably, such derivatives, analogs, and fragments retain theimmunomodulatory activity of the full-length, wild-type protein,polypeptide, or peptide.

Preferably, agents that are commercially available and known to functionas immunomodulatory agents are used in the methods of the invention. Theimmunomodulatory activity of an agent can be determined in vitro and/orin vivo by any technique well-known to one skilled in the art,including, e.g., by CTL assays, proliferation assays, and immunoassays(e.g. ELISAs) for the expression of particular proteins such asco-stimulatory molecules and cytokines.

5.6.2 Anti-Inflammatory Agents

Any anti-inflammatory agent, including agents useful in therapies forinflammatory disorders, well-known to one of skilled in the art can beused in accordance with methods of the invention to prevent, treat,manage, or ameliorate a RSV infection or one or more symptoms thereof.Non-limiting examples of anti-inflammatory agents include non-steroidalanti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs,anticholinergics (e.g., atropine sulfate, atropine methylnitrate, andipratropium bromide (ATROVENT™)), beta2-agonists (e.g., abuterol(VENTOLIN™ and PROVENTIL™), bitolterol (TORNALATE™), levalbuterol(XOPONEX™), metaproterenol (ALUPENT™), pirbuterol (MAXAIR™), terbutlaine(BRETHAIRE™ and BRETHINE™), albuterol (PROVENTIL™, REPETABS™, andVOLMAX™), formoterol (FORADIL AEROLIZER™), and salmeterol (SEREVENT™ andSEREVENT DISKUS™)), and methylxanthines (e.g., theophylline (UNIPHYL™,THEO-DUR™, SLO-BID™, and TEHO-42™)). Examples of NSAIDs include, but arenot limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac(VOLTAREN™), etodolac (LODINE™), fenoprofen (NALFON™), indomethacin(INDOCIN™), ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone(RELAFEN™), sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib(VIOXX™), naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) andnabumetone (RELAFEN™). Such NSAIDs function by inhibiting acyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examples of steroidalanti-inflammatory drugs include, but are not limited to,glucocorticoids, dexamethasone (DECADRON™), corticosteroids (e.g.,methylprednisolone (MEDROL™)), cortisone, hydrocortisone, prednisone(PREDNISONE™ and DELTASONE™), prednisolone (PRELONE™ and PEDIAPRED™),triamcinolone, azulfidine, and inhibitors of eicosanoids (e.g.,prostaglandins, thromboxanes, and leukotrienes (see Table 2, infra, fornon-limiting examples of leukotriene and typical dosages of suchagents)).

5.6.3 Anti-Viral Agents

Any anti-viral agent well-known to one of skilled in the art (inparticular, one useful for the treatment, prevention, management, oramelioration of a RSV infection) can be used in accordance with themethods of the invention to prevent, treat, manage or ameliorate a RSVinfection or one or more symptoms thereof. Non-limiting examples ofanti-viral agents include proteins, polypeptides, peptides, fusionproteins antibodies, nucleic acid molecules, organic molecules,inorganic molecules, and small molecules that inhibit and/or reduce theattachment of a virus to its receptor, the internalization of a virusinto a cell, the replication of a virus, or release of virus from acell. In particular, anti-viral agents include, but are not limited to,nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir,vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet,amantadine, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, and AZT.

In specific embodiments, the anti-viral agent is an antibody agent otherthan SYNAGIS® that is immunospecific for a viral antigen. As usedherein, the term “viral antigen” includes, but is not limited to, anyRSV peptide, polypeptide and protein (e.g., RSV F glycoprotein and RSV Gglycoprotein) that is capable of eliciting an immune response.

In preferred embodiments, the viral infection is RSV and the anti-viralantigen is an antibody other than SYNAGIS® that immunospecifically bindsto an antigen of RSV. In certain embodiments, the anti-RSV-antigenantibody immunospecifically binds to a RSV antigen of the Group A ofRSV. In other embodiments, the anti-RSV-antigen antibodyimmunospecifically binds to a RSV antigen of the Group B of RSV. Inother embodiments, the anti-RSV antigen antibody immunospecificallybinds to an antigen of RSV of one Group and cross reacts with theanalogous antigen of the other Group. In particular embodiments, theanti-RSV-antigen antibody immunospecifically binds to a RSVnucleoprotein, RSV phosphoprotein, RSV matrix protein, RSV smallhydrophobic protein, RSV RNA-dependent RNA polymerase, RSV F protein,and/or RSV G protein. In additional specific embodiments, theanti-RSV-antigen antibody binds to allelic variants of a RSVnucleoprotein, a RSV nucleocapsid protein, a RSV phosphoprotein, a RSVmatrix protein, a RSV attachment glycoprotein, a RSV fusionglycoprotein, a RSV nucleocapsid protein, a RSV matrix protein, a RSVsmall hydrophobic protein, a RSV RNA-dependent RNA polymerase, a RSV Fprotein, a RSV L protein, a RSV P protein, and/or a RSV G protein.

Anti-viral therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (57th ed., 2003).Additional information on respiratory viral infections is available inCecil Textbook of Medicine (18th ed., 1988).

5.7 Methods of Administering the SYNAGIS® Formulations

The invention provides methods of treatment, prophylaxis, andamelioration of a RSV infection or one or more symptoms thereof byadministrating to a subject of an effective amount of liquidformulations of the invention. The subject is preferably a mammal suchas non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey such as a cynomolgous monkey and a human). In apreferred embodiment, the subject is a human. In another preferredembodiment, the subject is a human infant or a human infant bornprematurely.

Various delivery systems are known and can be used to administer aliquid formulation of the present invention. Methods of administeringSYNAGIS® liquid formulations of the present invention include, but arenot limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epiduraladministration, topical administration, pulmonary administration, andmucosal administration (e.g., intranasal and oral routes). In a specificembodiment, liquid formulations of the present invention areadministered intramuscularly, intravenously, or subcutaneously and,preferably, intramuscularly. The formulations may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, pulmonary administration can beemployed, e.g., by use of an inhaler or nebulizer.

The invention also provides that a liquid formulation of the presentinvention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the SYNAGIS® orantigen-binding fragments thereof. Preferably, the liquid formulationsof the present invention are in a hermetically sealed containerindicating the quantity and concentration of the antibody or antibodyfragment. Preferably, the liquid formulation of the present invention issupplied in a hermetically sealed container at least 15 mg/ml, 20 mg/ml,30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml,100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml and, mostpreferably, 105 mg/ml, in a quantity of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml and, most preferably, 1.2ml.

The amount of the liquid formulations of the present invention whichwill be effective in the prevention, treatment, management oramelioration of a RSV infection or one or more symptoms thereof can bedetermined by standard clinical techniques. For example, the dosage ofthe composition which will be effective in the treatment, prevention oramelioration of symptoms associated with a RSV infection can bedetermined by administering the formulation to a cotton rat, measuringthe RSV titer after challenging the cotton rat with 10⁵ pfu of RSV andcomparing the RSV titer to that obtain for a cotton rat not administeredthe formulation. Accordingly, a dosage that results in a 2 log decreaseor a 99% reduction in RSV titer in the cotton rat challenged with 10⁵pfu of RSV relative to the cotton rat challenged with 10⁵ pfu of RSV butnot administered the formulation is the dosage of the formulation thatcan be administered to a human for prevention, treatment, management oramelioration of a RSV infection or one or more symptoms thereof. Thedosage of the formulation which will be effective in prevention,treatment, management or amelioration of a RSV infection or one or moresymptoms thereof can be determined by administering the formulation toan animal model (e.g., a cotton rat or monkey) and measuring the serumtiter of SYNAGIS® or antigen-binding fragments thereof. Accordingly, adosage of the formulation that results in a serum titer of at least 1μg/ml, preferably 2 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, atleast 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, at least 75 μg/ml,at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 200μg/ml, at least 250 μg/ml, at least 300 μg/ml, at least 350 μg/ml, atleast 400 μg/ml, or at least 450 μg/ml can be administered to a humanfor prevention, treatment, management or amelioration of a RSV infectionor one or more symptoms thereof. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the RSV infection,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model (e.g., thecotton rat or Cynomolgous monkey) test systems.

For antibodies (e.g., SYNAGIS®), proteins, polypeptides, peptides andfusion proteins, the dosage administered to a patient is typically about1 mg/kg to 30 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 10 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 15 mg/kg of the patient's bodyweight. Generally, human antibodies have a longer half-life within thehuman body than antibodies from other species due to the immune responseto the foreign polypeptides. Thus, lower dosages of human antibodies andless frequent administration is often possible. Further, the dosage,volume and frequency of administration of liquid formulations of thepresent invention may be reduced by increasing the concentration ofSYNAGIS® or an antigen-binding fragment thereof in the formulations,increasing affinity and/or avidity of SYNAGIS® or an antigen-bindingfragment thereof, and/or increasing the half-life of SYNAGIS® or anantigen-binding fragment thereof.

Exemplary doses of a small molecule include milligram or microgramamounts of the small molecule per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram).

In a specific embodiment, a mammal, preferably a human, is administereda stable liquid formulation of the present invention for the prevention,treatment, management or amelioration of a RSV infection or one or moresymptoms thereof in an amount effective for decreasing RSV titers. Inaccordance with this embodiment, an effective amount of the liquidformulations of the present invention reduces the RSV titers in the lungas measured, for example, by the concentration of RSV in sputum samplesor a lavage from the lungs from a mammal. In another embodiment, amammal, preferably a human, is administered a liquid formulation of thepresent invention for the prevention, treatment, management oramelioration of a RSV infection or one or more symptoms thereof in anamount effective for inducing an immune response in the mammal.

In another embodiment, a mammal, preferably a human, is administered afirst dose of a liquid formulation of the present invention comprising30 mg/kg or less, 15 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 3mg/kg or less, 1 mg/kg or less, or 0.5 mg/kg or less of SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management or amelioration of a RSV infection or one or more symptomsthereof in an amount effective to induce a serum titer of at least 1μg/ml, preferably at least 2 μg/ml, at least 5 μg/ml, at least 10 μg/ml,at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30μg/ml, at least 35 μg/ml, at least 40 μg/ml 20 days (preferably 25, 30,35, 40 days) after the administration of the first dose and prior to theadministration of a subsequent dose. In a specific embodiment, a liquidformulation of the present invention comprises SYNAGIS® or anantigen-binding fragment thereof and is administered to a subject afirst dose of about 1 mg/kg to about 30 mg/kg to induce a serum titer ofabout 40 μg/ml or higher 30 days after the administration of the firstdose and prior to the administration of a subsequent dose. Preferably,the serum titer of said SYNAGIS® or an antigen-binding fragment thereofis less than 50 μg/ml 30 days after the administration of the first doseand prior to the administration of a subsequent dose.

In another embodiment, a mammal, preferably a human, is administered afirst dose of a liquid formulations of the present invention comprising30 mg/kg or less, 15 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 3mg/kg or less, 1 mg/kg or less or 0.5 mg/kg or less of SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management or amelioration of a RSV infection or one or more symptomsthereof in an amount effective to induce a serum titer of at least 1μg/ml, preferably at least 2 μg/ml, at least 5 μg/ml, at least 10 μg/ml,at least 15 μg/ml, at least 20 μg/ml, or at least 25 μg/ml 20 days(preferably 25, 30, 35, 40 days) after the administration of the firstdose and prior to the administration of subsequent dose. Preferably, theserum titer of said SYNAGIS® or an antigen-binding fragment thereof isless than 30 μg/ml 30 days after the administration of the first doseand prior to the administration of a subsequent dose.

In another embodiment, a mammal, preferably a human, is administered afirst dose of a liquid formulation of the present invention comprising30 mg/kg or less, 15 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1mg/kg or less or 0.5 mg/kg or less of a modified form of SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management or amelioration of a RSV infection or one or more symptomsthereof which has an increased in vivo half-life in an amount effectiveto induce a serum titer of at least 1 μg/ml, preferably at least 2μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least20 μg/ml, or at least 25 μg/ml 25 days (preferably 30, 35, or 40 days)after the administration of the first dose and prior to theadministration of a subsequent dose. Preferably, the serum titer of saidSYNAGIS® or an antigen-binding fragment thereof is less than 30. [g/ml30 days after the administration of the first dose and prior to theadministration of a subsequent dose.

In another embodiment, a mammal, preferably a human, is administered afirst dose of a liquid formulation of the present invention comprising30 mg/kg or less, 15 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1mg/kg or less, or 0.5 mg/kg or less of a modified SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management or amelioration of a RSV infection or one or more symptomsthereof which has an increased in vivo half-life in an amount effectiveto induce a serum titer of at least 1 μg/ml, preferably at least 2μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least20 μg/ml, or at least 25 μg/ml 25 days (preferably 30, 35, or 40 days)after the administration of the first dose and prior to theadministration of a subsequent dose. Preferably, the serum titer of saidSYNAGIS® or an antigen-binding fragment thereof is less than 30 μg/ml 30days after the administration of the first dose and prior to theadministration of a subsequent dose.

In another embodiment, a mammal, preferably a human, is administered afirst dose of the liquid formulation comprising approximately 30 mg/kgor less, 15 mg/kg or less (preferably 10 mg/kg or less, 5 mg/kg or less,3 mg/kg or less, 1 mg/kg or less, or 0.5 mg/kg or less) of a modifiedform of SYNAGIS® or an antigen-binding fragment thereof which has anincreased in vivo half-life for the prevention, treatment, management oramelioration of a RSV infection or one or more symptoms thereof in anamount effective to induce a serum titer of at least 30 μg/ml,preferably at least 35 μg/ml, at least 40 μg/ml, or at least 50 μg/ml 25days (preferably 30, 35, or 40 days) after the administration of thefirst dose and prior to the administration of a subsequent dose.

In one embodiment, a mammal, preferably a human, is administered a firstdose of a liquid formulation of the present invention for pulmonarydelivery comprising 30 mg/kg or less, 15 mg/kg or less, 5 mg/kg or less,3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.01 mg/kg orless of SYNAGIS® or an antigen-binding fragment thereof for theprevention, treatment, management or amelioration of a RSV infection orone or more symptoms thereof in an amount effective to induce a titer ofat least 20 ng per mg of lung protein (preferably at least 40 ng/mg, atleast 60 ng/mg, at least 80 ng/mg, at least 50 ng/mg, at least 75 ng/mg,at least 100 ng/mg, or at least 150 ng/mg) in an intubation sample orlavage from the lungs of said mammal 20 days (preferably 25, 30, 35, or40 days) after the administration of the first dose and prior to theadministration of a subsequent dose. Preferably, the serum titer of saidSYNAGIS® or an antigen-binding fragment thereof is less than 100 ng/mlof protein 30 days after the administration of the first dose and priorto the administration of a subsequent dose.

In another embodiment, a mammal, preferably a human, is administered afirst dose of a liquid formulation of the present invention 10 mg/kg orless, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, or 0.5 mg/kg orless of SYNAGIS® or an antigen-binding fragment thereof for theprevention, treatment, management or amelioration of a RSV infection orone or more symptoms thereof in an amount effective to induce a serumtiter of at least 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, atleast 80 μg/ml, at least 100 μg/ml, at least 120 μg/ml, at least 150μg/ml, at least 200 μg/ml, at least 250 μg/ml, or at least 300 μg/ml 20days (preferably 25, 30, 35 or 40 days) after the administration of thefirst dose. In another embodiment, a mammal, preferably a human, isadministered a first dose of a liquid formulation of the presentinvention comprising approximately 15 mg/kg of SYNAGIS® or anantigen-binding fragment thereof for the prevention, treatment,management or amelioration of a RSV infection or one or more symptomsthereof in an amount effective to induce a serum titer of at least 100μg/ml, at least 125 :g/ml, at least 150 μg/ml, at least 200 μg/ml, atleast 250 μg/ml, at least 300 μg/ml, at least 350 μg/ml, at least 400μg/ml, or at least 450 :g/ml 20 days (preferably 25, 30, 35 or 40 days)after the administration of the first dose. The term “approximately 15mg/kg” as used herein refers to a range of between 14 mg/kg and 16mg/kg.

In another embodiment, a mammal, preferably a human, is administered adose of a liquid formulation of the present invention comprisingSYNAGIS® or an antigen-binding fragment thereof for the prevention of aRSV infection or a symptom thereof in an amount effective to induce aprophylactically effective serum titer of less than 10 μg/ml, less than8 μg/ml, less than 5 μg/ml, less than 3 μg/ml, less than 1 μg/ml, orless than 0.5 μg/ml 30 days after the administration of the dose,wherein said prophylactically effective serum titer is the serum titerthat reduces the incidence of RSV infection in the human or the serumtiter in a cotton rat that results in a RSV titer 5 days after challengewith 10⁵ pfu RSV that is 99% lower than the RSV titer in the cotton rat5 days after challenge with 10⁵ pfu of RSV in a cotton rat notadministered the dose prior to challenge. Preferably, the dose of thetherapeutic or pharmaceutical composition comprises 10 mg/kg or less, 5mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, or 0.5 mg/kg or less ofSYNAGIS® or an antigen-binding fragment thereof.

In yet another embodiment, a mammal, preferably a human, is administereda dose of a liquid formulation of the present invention comprisingSYNAGIS® or an antigen-binding fragment thereof for the treatment,management or amelioration of a RSV infection or one or more symptomsthereof in an amount effective to induce a therapeutically effectiveserum titer of less than 10 μg/ml, less than 8 μg/ml, less than 5 μg/ml,less than 3 μg/ml, less than 1 μg/ml, or less than 0.5 μg/ml 30 daysafter the administration of the dose, wherein said therapeuticallyeffective serum titer is the serum titer that reduces the severity orlength of RSV infection or is the serum titer in a cotton rat thatresults in a RSV titer in the rat 5 days after challenge with 10⁵ pfuRSV that is 99% lower than the RSV titer 5 days after challenge with 10⁵pfu of RSV in a cotton rat not administered the dose prior to challenge.Preferably, the dose of the liquid formulation of the present inventioncomprises 12 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 3 mg/kgor less, 1 mg/kg or less, or 0.5 mg/kg or less of an antibody or anantigen-binding fragment thereof.

In a specific embodiment, formulations of the present invention areadministered once a month just prior to or during the RSV season. Inanother embodiment, the formulations are administered every two monthsjust prior to or during the RSV season. In yet another embodiment, thestable formulations of the present invention are administered once justprior to or during the RSV season. The term “RSV season” refers to theseason when RSV infection is most likely to occur. Typically, the RSVseason in the northern hemisphere commences in November and laststhrough April.

In one embodiment, a liquid formulation comprising approximately 5 mg/kgor less (preferably 1.5 mg/kg or less) of SYNAGIS® or an antigen-bindingfragment thereof is administered five times, 3 times, or 1 to 2 timesduring a RSV season to a mammal, preferably a human. In anotherembodiment, approximately 1.5 mg/kg of SYNAGIS® or an antigen-bindingfragment thereof, in the liquid formulations of the present invention isadministered monthly five times during a RSV season to a mammal,preferably a human, intramuscularly. In another embodiment, 3 mg/kg ofSYNAGIS® or an antigen-binding fragment thereof in the liquidformulation of the invention is administered monthly three times duringa RSV season to a mammal, preferably a human, intramuscularly. In yetanother embodiment, 5 mg/kg of an SYNAGIS® or an antigen-bindingfragment thereof in a liquid formulation of the invention isadministered monthly one to two times during a RSV season to a mammal,preferably a human, intramuscularly.

In a specific embodiment, 15 mg/kg of SYNAGIS® or an antigen-bindingfragment thereof in the liquid formulation of the present invention isadministered to a mammal, preferably a human, intramuscularly five timesduring a RSV season, wherein said SYNAGIS® or antibody fragment has anincreased in vivo half-life. In another embodiment, approximately 5mg/kg or less (preferably 1.5 mg/kg or less) of SYNAGIS® or anantigen-binding fragment thereof in the liquid formulation of thepresent invention is administered five times, 3 times, or 1 to 2 timesduring a RSV season to a mammal, preferably a human. In anotherembodiment, 3 mg/kg of SYNAGIS® or an antigen-binding fragment thereof,which has an increased in vivo half-life, in the liquid formulation ofthe present invention is administered monthly three times during a RSVseason to a mammal, preferably a human, intramuscularly. In anotherembodiment, 5 mg/kg of SYNAGIS® or an antigen-binding fragment thereof,which has an increased in vivo half life, in the liquid formulation ofthe present invention is administered to a mammal, preferably a human,intramuscularly twice times during a RSV season.

5.8 Biological Assays

5.8.1 Immunospecificity of the Antibodies of the Invention

Antibodies of the present invention or fragments thereof may becharacterized in a variety of ways well-known to one of skill in theart. In particular, antibodies of the invention or antigen-bindingfragments thereof in a liquid formulation of the present invention maybe assayed for the ability to immunospecifically bind to an epitope of arespiratory syncytial virus. Such an assay may be performed in solution(e.g., Houghten, 1992, Bio/Techniques 13:412–421), on beads (Lam, 1991,Nature 354:82–84), on chips (Fodor, 1993, Nature 364:555–556), onbacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), on plasmids (Cull et al., 1992, Proc. Natl.Acad. Sci. USA 89:1865–1869) or on phage (Scott and Smith, 1990, Science249:386–390; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA87:6378–6382; and Felici, 1991, J. Mol. Biol. 222:301–310) (each ofthese references is incorporated herein in its entirety by reference).SYNAGIS® or an antigen-binding fragment thereof in a liquid formulationof the present invention can be assayed for its specificity andaffinity.

SYNAGIS® or an antigen-binding fragment thereof of the present inventionmay be assayed for immunospecific binding to a RSV antigen andcross-reactivity with other antigens by any method known in the art.Immunoassays which can be used to analyze immunospecific binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well-known in the art (see, e.g., Ausubel etal., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety).

5.8.2 In Vitro and In Vivo Assays

A liquid formulation or a combination therapy of the present inventioncan be tested in vitro and/or in vivo in various assays or suitableanimal model systems for its activity.

A liquid formulation of the present invention for treating, managing,preventing, or ameliorating a RSV infection or one or more symptomsthereof can be tested for its ability to inhibit viral replication orreduce viral load in in vitro assays. For example, viral replication canbe assayed by a plaque assay such as described, e.g., by Johnson et al.,1997, Journal of Infectious Diseases 176:1215–1224 176:1215–1224. Aliquid formulation of the invention administered according to themethods of the invention can also be assayed for their ability toinhibit or down-regulate the expression of viral polypeptides.Techniques known to those of skill in the art, including, but notlimited to, western blot analysis, northern blot analysis, and RT-PCRcan be used to measure the expression of viral polypeptides and/or viraltiters.

A liquid formulation of the invention can be tested in suitable animalmodel systems prior to use in humans. Such animal model systems include,but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs,rabbits, etc. Any animal system well-known in the art may be used.Several aspects of the procedure may vary; said aspects include, but arenot limited to, the temporal regime of administering the therapies(e.g., prophylactic and/or therapeutic agents) whether such therapiesare administered separately or as an admixture, and the frequency ofadministration of the therapies.

Animal models can be used to assess the efficacy of the methods of theinvention for treating, managing, preventing, or ameliorating a RSVinfection or one or more symptom thereof. Animal models for RSVinfection include, but are not limited to, those as described by, e.g.,Piedimonte et al., Am J Physiol 1999, 277:L831–L840; McArthur-Vaughan etal., J. Med. Primatol. 2002, 31(2):61–73; and Byrd et al., Clin. Infect.Dis. 1997, 25(6):1363–8. In a specific embodiment, cotton rats areadministered a liquid formulation comprising SYNAGIS® according to themethods of the invention, challenged with 10⁵ pfu of RSV, and four ormore days later, the rats are sacrificed and RSV titer and SYNAGIS®serum titer is determined. Accordingly, a dosage that results in a 2 logdecrease or a 99% reduction in RSV titer in the cotton rat challengedwith 10⁵ pfu of RSV relative to the cotton rat challenged with 10⁵ pfuof RSV but not administered the formulation is the dosage of theformulation that can be administered to a human for the treatment,prevention or amelioration of a RSV infection or one or more symptomsthereof. Further, this embodiment, the tissues (e.g., the lung tissues)from the sacrificed rats can be examined for histological changes.

The administration of a liquid formulation of the invention according tothe methods of the present invention can be tested for its ability todecrease the time course of a RSV infection by at least 25%, preferablyat least 50%, at least 60%, at least 75%, at least 85%, at least 95%, orat least 99%. A liquid formulation of the invention can also be testedfor its ability to increase the survival period of humans suffering froma RSV infection by at least 25%, preferably at least 50%, at least 60%,at least 75%, at least 85%, at least 95%, or at least 99%. Further, aliquid formulation of the invention can be tested for its ability reducethe hospitalization period of a human suffering from RSV infection by atleast 60%, preferably at least 75%, at least 85%, at least 95%, or atleast 99%. Techniques known to those of skill in the art can be used toanalyze the function of a liquid formulation of the invention in vivo.

Further, any in vitro or in vivo assays known to those skilled in theart can be used to evaluate the prophylactic and/or therapeutic utilityof a liquid formulation of the invention disclosed herein for a RSVinfection or one or more symptoms thereof.

5.8.3 Toxicity Assays

The toxicity and/or efficacy of the prophylactic and/or therapeuticprotocols of the instant invention can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Therapies that exhibit large therapeutic indices are preferred. Whiletherapies that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such agents to the siteof affected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any therapy used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

5.9 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with a liquid formulation of the invention forthe prevention, treatment, management or amelioration of a RSV infectionor one or more symptoms thereof. In a specific embodiment, the liquidformulations of the invention comprise SYNAGIS® or an antigen-bindingfragment thereof recombinantly fused or chemically conjugated to anothermoiety, including but not limited to, a heterologous protein, aheterologous polypeptide, a heterologous peptide, a large molecule, asmall molecule, a marker sequence, a diagnostic or detectable agent, atherapeutic moiety, a drug moiety, a radioactive metal ion, a secondantibody, and a solid support.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises a liquid formulation of theinvention, in one or more containers. In another embodiment, a kitcomprises a liquid formulation of the invention, in one or morecontainers, and one or more other prophylactic or therapeutic agentsuseful for the prevention, management or treatment of a RSV infection orone or more symptoms thereof, in one or more other containers.Preferably, the kit further comprises instructions for preventing,treating, managing or ameliorating a RSV infection (e.g., using theliquid formulations of the invention alone or in combination withanother prophylactic or therapeutic agent), as well as side effects anddosage information for method of administration. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

6. EXAMPLES 6.1 Example 1 Stability Study

An antibody formulation of the present invention comprising, in anaqueous carrier, 25 mM of histidine, 1.6 mM of glycine, and SYNAGIS® atpH 6 was prepared according to the following protocol:

For a 1 kg solution of buffer: In 800 g water, 3.875 g histidine (freebase) and 0.12 g glycine were dissolved. The pH was adjusted with 6 NHCl to 6.0±0.2. Water was added to bring the total mass up to 1.0 kg(qs).

For the diafiltration: After the chromatography steps in thepurification process, SYNAGIS® was concentrated to a target of 150 g/L.The concentrated product is diafiltered into formulation buffer. Theformulated product was diluted to a target manufacturing concentrationof 103±3 g/L.

For a stability study, two formulations were prepared: one contained 105mg/ml of SYNAGIS® and the other contained 160 mg/ml of SYNAGIS®. Thestability of each formulation was measured using HPSEC in terms ofdegrees of aggregate formation and fragmentation during the storage at2–8° C. for up to 15 months and at 38–42° C. for up to 1 year. For theHPSEC analysis, typically, TosoHaas G3000WXL column with a mobile phasecontaining 0.1 M sodium phosphate and 0.1 M sodium sulfate, pH 6.8, isused at a flow rate of 0.8 ml/min. A sample containing 250 mg of proteinin an appropriate volume is injected into the column and protein peaksare detected by 280 nm UV and/or fluorescence (280 nm excitation and 340nm emission).

The data showed that there was no detectable increase in aggregationwhen each formulation of SYNAGIS® was stored at 2–8° C. for 15 months asshown in Table 3.

TABLE 3 Percent Aggregates during Storage at 2–8° C. % Aggregates Month105 mg/ml 160 mg/ml 0 0.3 0.4 5 0.3 0.3 8 0.4 — 12 0.4 — 15 0.4 0.5±0.1% error.

When the formulations were stored at 38–42° C. for 60 days, about 1.5%increase in aggregate was observed with the formulation containing 105mg/ml of SYNAGIS® and 2.0% increase was observed with the formulationcontaining 160 mg/ml of SYNAGIS®.

6.2 Example 2 Clinical Study

The liquid formulation of the present invention comprising 100 mg/ml ofSYNAGIS® in an aqueous solution containing 25 mM of histidine and 1.6 mMof glycine at pH 6 is tested for safety and tolerability study in aPhase I, parallel group, double-blind, randomized study at two sites.The study drugs are a liquid (Liq) formulation of SYNAGIS® and thecurrently licensed lyophilized (Lyo) formulation of SYNAGIS®. A total of48 volunteers will be randomized to one of four treatment groups:

GROUP 1: N = 12  3 mg/kg SYNAGIS ® (Liq) at Study Days 0 and 30 (IM)GROUP 2: N = 12  3 mg/kg SYNAGIS ® (Lyo) at Study Days 0 and 30 (IM)GROUP 3: N = 12 15 mg/kg SYNAGIS ® (Liq) at Study Day 0 (IV) GROUP 4: N= 12 15 mg/kg SYNAGIS ® (Lyo) at Study Day 0 (IV)

Vital signs will be obtained before and 30 minutes after each dose ofstudy drug. Adverse events will be monitored through 30 days after thelast dose of study drug and serious adverse events will be monitoredthrough Study Day 60.

On Study Day 0, all volunteers will have blood collected for SYNAGIS®serum concentration before dosing, at the end of infusion (IV dosegroups only), and at 0.25, 0.5, 1, 4, 8, and 12 hours after IM injectionor end of infusion. Subsequently, blood samples for determination ofSYNAGIS® levels will be collected daily though Study Day 5 and on StudyDays 7, 14, 21, 30, 37 (IM groups only), and 60. Serum samples foranti-SYNAGIS® antibodies will be collected on Study Days 0, 7, 14, 21,30, 37 (IM groups only), and 60. Samples for serum chemistry and CBCwith differential and platelets, and urine samples for urinalysis willbe collected on Study Day 0 as well as 7 days after each dose ofSYNAGIS® (Study Day 7 for the IV groups and Study Days 7 and 37 for theIM groups). Urine βHCG tests will be performed on the day of dosingbefore each dose of SYNAGIS® (Study Day 0 for the IV groups and StudyDays 0 and 30 for the IM groups). A study flow diagram is shown in FIG.2.

6.2.1 Study Procedures

A. Volunteer Selection

The volunteers in this study will be healthy male or female adults. Thevolunteer will be counseled by an investigator (physician) who willaddress the questions and concerns of the volunteer and secure writteninformed consent for participation in the study. Written informedconsent will be obtained prior to conducting study procedures oradministration of study drug.

a. Inclusion Criteria

Volunteers must meet all of the following criteria:

-   -   1. Male or female.    -   2. Age 18 through 49 years at the time of the first dose of        study drug.    -   3. Weight≦150 kg.    -   4. Written informed consent obtained from the volunteer.

5. Sexually active females, unless surgically sterile, must have used aneffective method of avoiding pregnancy (including oral or implantedcontraceptives, IUD, female condom, diaphragm with spermicide, cervicalcap, abstinence, use of a condom by the sexual partner or sterile sexualpartner) for 14 days prior to the first dose of study drug, must agreeto continue using such precautions for 30 days after the final dose ofstudy drug, and must have a negative serum pregnancy test within 2 daysprior to the first dose of study drug.

-   -   6. Healthy by medical history and physical examination.    -   7. Ability to complete follow-up period of 60 days as required        by the protocol.

b. Exclusion Criteria

Volunteers must have none of the following:

-   -   1. Acute illness at the start of the study    -   2. Fever≧99.5° F. at the start of the study    -   3. Any drug therapy within 7 days prior to Study Day 0 (except        for contraceptives)    -   4. Blood donation in excess of 400 mL within 6 months of study        start    -   5. Receipt of immunoglobulin or blood products within 60 days        before entry into the study    -   6. Receipt of any investigational drug therapy or standard        vaccine within 120 days before the first dose of study drug in        this protocol through 60 days after the final dose of study drug    -   7. History of immunodeficiency    -   8. History of allergic disease or reactions likely to be        exacerbated by any component of the study drug    -   9. Previous medical history or evidence of an intercurrent        illness that may compromise the safety of the volunteer in the        study    -   10. Evidence of infection with hepatitis A, B, or C virus or        HIV-1    -   11. At screening (must be within 21 days before entry into the        study) any of the following: CBC: Hgb<12.0 gm/dl; WBC<4,000/mm³;        platelet count<120,000/mm³ (or laboratory normal values); AST,        ALT, BUN, creatinine>upper limit of normal; other abnormal        laboratory values in the screening panel which in the opinion of        the principal investigator are judged to be clinically        significant; other abnormal laboratory values in the screening        panel which in the opinion of the principal investigator are        judged to potentially confound analysis of study results    -   12. Nursing mother    -   13. History of alcohol or drug abuse within the past 2 years    -   14. Evidence of any systemic disease on physical examination

B. Randomization

a. Volunteer Randomization Procedures and Treatment Allocation

At the screening visit, volunteers will be evaluated by the principalinvestigator to assess eligibility for entry into the study. A masterlog will be maintained for all screened volunteers. Volunteers who signan informed consent and who meet eligibility criteria will be enteredinto the study. When a volunteer arrives in the study site forrandomization (Study Day 0), the investigator will confirm that thevolunteer meets all inclusion and exclusion criteria. The investigatorwill then assign a patient identification number (PID). Patientidentification numbers will be assigned sequentially within each of thetwo study sites beginning with #101 in site 1 and with #201 in site 2.Volunteers will be considered to have entered the study when the PID isassigned. A randomization list provided to the study pharmacist at eachstudy site will contain assignments to each of the four treatment groupsfor volunteers at that site. The investigator will notify MedImmune byfacsimile transmission (fax) at 301-527-4217 that a volunteer has beenrandomized. Volunteers who have been assigned a PID and do not receiveany study drug, who receive an incomplete infusion of study drug, who donot receive both IM injections of study drug, or who do not complete atleast 50% of the study visits may be replaced at the discretion of thesponsor. Volunteers who withdraw due to an adverse event or whose statuscannot be ascertained will not be replaced. Volunteers who withdrawconsent for reasons other than an adverse event may be replaced.Replacement volunteers will be assigned a new PID. Volunteers who arereplaced will continue to be followed for safety according to theprotocol.

b. Blinding

This is a double-blind study. Blinding will be maintained for assignmentof volunteers to lyophilized or liquid formulation within IM or IVgroups. In order to maintain blinding during administration of the twoformulations of SYNAGIS®, the study pharmacist at each site will preparethe study drug at a site physically removed from the treatment stationand shielded from the observation of the principal investigator or anystudy personnel directly involved in the conduct of the study. For IMinjection, the pharmacist will prepare identical appearing 5 mL syringescontaining the calculated volume of either liquid or reconstitutedlyophilized SYNAGIS®. For IV infusion, the pharmacist will prepareidentical appearing 200 mL infusion bags containing the calculatedvolume of either liquid or reconstituted lyophilized SYNAGIS®. Labelswill not identify whether the syringe/bag contains liquid orreconstituted lyophilized SYNAGIS®. An independent monitor who willreview the pharmacy record only, the statistician and clinical suppliesmanager at MedImmune, and the study pharmacist at the study site are theonly individuals who will have access to the randomization list whichidentifies a volunteer's study treatment allocation. These individualsmust not reveal randomization information to anyone. In the event thatthe study treatment for a volunteer becomes known to the investigator,MedImmune must be notified immediately by the investigator. Allinstances of unblinding will be documented in the study report.

C. Study Drug

a. Study Drug Supplies and Accountability

The sponsor will provide the investigator with adequate quantities ofliquid SYNAGIS®, lyophilized SYNAGIS®, and diluent (sterile water forinjection). Study drug should be stored at 2° C. to 8° C. (36° F. to 46°F.) and must not be frozen.

Liquid SYNAGIS® will be provided in 3 mL vials containing 100 mg ofsterile liquid product in a volume of 1 mL (25 mM histidine, 1.6 mMglycine, at pH 6.0).

Lyophilized SYNAGIS® will be provided in 5 mL vials containing 100 mg ofsterile lyophilized product which when formulated (beforelyophilization) contains 25 mM histidine, 1.6 mM glycine, and 3% (w/v)mannitol at pH 6.0.

The study pharmacist is required to maintain accurate drugaccountability records. Upon completion of the study, all study drugaccountability records will be returned to the sponsor. All unused studydrug will be returned to the sponsor.

b. Treatment Regimens

GROUP 1: N = 12  3 mg/kg SYNAGIS ® (Liq) at Study Days 0 and 30 (IM)GROUP 2: N = 12  3 mg/kg SYNAGIS ® (Lyo) at Study Days 0 and 30 (IM)GROUP 3: N = 12 15 mg/kg SYNAGIS ® (Liq) at Study Day 0 (IV) GROUP 4: N= 12 15 mg/kg SYNAGIS ® (Lyo) at Study Day 0 (IV)

c. Ordering and Preparation of Study Drug

The dose of study drug for administration must be prepared by the studypharmacist. The study drug prescription form indicating the PID and thevolunteer's body weight will be sent to the pharmacist by theinvestigator (or designee). The study pharmacist will then use thisinformation to prepare the study drug.

To prepare liquid or lyophilized SYNAGIS® for administration, thepharmacist should remove the tab portion of the vial cap and clean therubber stopper with 70% ethanol or equivalent. The vial should bevented. Doses for each volunteer will be calculated as described belowbased on the volunteer's weight (to the nearest 0.1 kilogram) on the daySYNAGIS® is administered. The dose should be rounded to the nearest 0.1mL. All preparations of study drug must be administered within 6 hoursafter entering the vial of SYNAGIS®. If it is not administered within 6hours a new vial or vials must be used.

Preparation of Liquid SYNAGIS®

(1) For IM injections: The required volume of liquid SYNAGIS® (100mg/mL) will be obtained by pooling the contents of as many vials asnecessary with a 5 mL syringe.Dose (mL)=[Volunteer Weight (kg)×Dose Level (3 mg/kg)]÷DrugConcentration (100 mg/mL)

-   -   Example: A volunteer who weighs 75.6 kg receives 2.3 mL of        SYNAGIS® (75.6 kg×3 mg/kg)÷100 mg/mL=2.268 mL (rounded to 2.3        mL)

(2) For IV infusions: The required volume of liquid SYNAGIS® (100 mg/mL)will be obtained by pooling the contents of as many vials as necessarywith a 20 mL (or larger) syringe.Dose (mL)=[Volunteer Weight (kg)×Dose Level (15 mg/kg)]÷DrugConcentration (100 mg/mL)

This volume of liquid SYNAGIS® will then be injected into an empty 200mL infusion bag and diluted with diluent 1:4 by adding four volumes ofdiluent to the bag for a final concentration of 20 mg/mL SYNAGIS®.

-   -   Example: A volunteer who weighs 71.4 kg receives 10.7 mL of        SYNAGIS® [(71.4 kg×15 mg/kg)+100 mg/mL=10.71 mL (rounded to 10.7        mL)] and 42.8 mL of diluent (4×10.7) for a total infusion volume        of 53.5 mL.        Preparation of Lyophilized SYNAGIS®

(1) For IM injections: One (1) ml of diluent should be added slowly tothe vial of lyophilized SYNAGIS® for a final concentration of 100 mg/mLSYNAGIS®. The vial should then be gently swirled for 30 seconds to avoidfoaming. Reconstituted SYNAGIS® should stand at room temperature for aminimum of 20 minutes until SYNAGIS® clarifies. The required volume ofreconstituted lyophilized SYNAGIS® (100 mg/mL) will be obtained bypooling the contents of as many vials as necessary with a 5 mL syringe.Dose (mL)=[Volunteer Weight (kg)×Dose Level (3 mg/kg)]÷DrugConcentration (100 mg/mL)

-   -   Example: A volunteer who weighs 75.6 kg receives 2.3 mL of        SYNAGIS® (75.6 kg×3 mg/kg)÷100 mg/mL=2.268 mL (rounded to 2.3        mL)

(2) For IV infusions: Five (5) ml of diluent should be added slowly tothe vial of lyophilized SYNAGIS® for a final concentration of 20 mg/mLSYNAGIS®. The vial should then be gently swirled for 30 seconds to avoidfoaming. Reconstituted SYNAGIS® should stand at room temperature for aminimum of 20 minutes until SYNAGIS® clarifies. The required volume ofreconstituted lyophilized SYNAGIS® (20 mg/mL) will be obtained bypooling the contents of as many vials as necessary with a 20 mL (orlarger) syringe and injecting this volume into an empty 200 mL infusionbag.Dose (mL)=[Volunteer Weight (kg)×Dose Level (15 mg/kg)]÷DrugConcentration (20 mg/mL)]

-   -   Example: A volunteer who weighs 71.4 kg receives 53.6 mL of        SYNAGIS® (71.4 kg×15 mg/kg)÷20 mg/mL=53.55 mL (rounded to 53.6        mL)

d. Administration of Study Drug

Study drug for the IM and IV treatment groups will be dispensed from thepharmacy in identical appearing 5 mL syringes and identical appearing200 mL infusion bags, respectively.

IM Injection

The study drug will be administered by IM injection into the deltoidmuscle (after confirming the needle is not in a blood vessel) usingstandard aseptic technique. Volunteers will remain under observation inthe study site for at least 30 minutes after the injection.

IV Infusion

Prior to drug administration, an IV catheter will be placed in anaccessible vein using standard insertion techniques. Patency of the IVcatheter will be maintained by a continuous IV infusion of 5% Dextrosefor Injection USP at a rate of 10 to 25 mL/h. The dextrose infusion willbe interrupted, and SYNAGIS® will be infused through a low proteinbinding 0.22 μm filter at a rate of approximately 1–2 mL/minute. AfterSYNAGIS® has been administered, the IV tubing should be flushed and keptopen with 5% Dextrose for Injection USP at 10 to 25 mL/h until the IVcatheter is removed.

e. Concomitant Medications

All concomitant medications used by the volunteer from Study Day 0through Study Day 60 will be recorded on the case report form.Volunteers may not receive the following:

-   -   1. Immunosuppressive medication (inhaled and topical        corticosteroids are permitted).    -   2. Investigational agents from 120 days before study entry        through Study Day 60.

The sponsor must be notified if any volunteer receives prohibitedconcomitant medications. Volunteers may receive medications to treatadverse events as deemed necessary by the investigator or thevolunteer's physician.

D. Schedule of Volunteer Evaluations

All volunteers who are assigned a PID and receive any study drug will befollowed according to the protocol regardless of the number of doses ofstudy drug received, unless consent for follow-up is withdrawn. Thesponsor must be notified of all deviations from protocol visits orevaluations and these evaluations, if applicable, must be rescheduled orperformed at the nearest possible time to the original schedule.

Volunteers will be instructed to call study personnel to report anyabnormalities during the intervals between study visits and to come tothe study site if medical evaluation is needed and the urgency of thesituation permits. For emergency and other unscheduled visits to amedical facility other than the study site, medical records will beobtained by the investigator. A schedule of screening and on-study visitprocedures is presented in Table 4, followed by a detailed descriptionof each visit.

TABLE 4 Schedule of Volunteer Evaluations Study Day Scn 0 1 2 3 4 5 7 1421 30 37 60 Written Informed Consent x Verify Eligibility Criteria x xMedical History x Physical Examination x Height and body weight^(g) x xx^(d) Urinalysis x x X x^(d) Hepatitis A, B, C x Serum βHCG^(b) x UrineβHCG^(b) x x^(d) Serum Chemistry^(c) x x X x^(d) CBC, Differential,Platelets x x X x^(d) SYNAGIS ® Serum Level X^(a) x x x x x X x x xx^(d) x Anti-SYNAGIS ® Antibody x X x x x x^(d) x Vital Signs^(e) xx^(d) Randomization/Assignment x of PID Study Drug Injection (IM x x^(d)Groups) Study Drug Infusion (IV x Groups) Assessment of Adverse x x x xx x X x x x x^(d) x Events^(f)

-   a. Blood will be sampled for SYNAGIS® serum concentration before    dosing, at the end of infusion (IV dose groups only), and at 0.25,    0.5, 1, 4, 8, and 12 hours after injection/end of infusion.-   b. Females only.-   c. ALT, AST, BUN, Creatinine.-   d. IM dose groups only.-   e. Vital signs obtained before and 30 minutes after each dose.-   f. Adverse events through 30 days after each dose. Serious adverse    events through Study Day 60.-   g. Body weight only at Study Day 30.

a. Screening

Note: All screening laboratory assessments must be performed within 21days before study entry (Study Day 0). The screening evaluations may becarried out over more than one visit.

-   -   1. Written informed consent    -   2. Verify eligibility criteria    -   3. Screening medical history    -   4. Screening physical examination    -   5. Height and body weight    -   6. Urinalysis    -   7. Blood collection for screening        -   Serum for hepatitis A antibody, hepatitis B surface antigen,            hepatitis C antibody        -   Serum βHCG (female volunteers only)        -   Chemistry panel (AST, ALT, BUN, creatinine)        -   CBC with differential and platelet count            Study Day 0: Dose 1            Visit 1    -   1. Verify eligibility criteria    -   2. Height and body weight    -   3. Urinalysis    -   4. Urine βHCG (female volunteers only)    -   5. Baseline blood collection        -   Chemistry panel (AST, ALT, BUN, creatinine)        -   CBC with differential and platelet count        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS® antibody    -   6. Randomization and assignment of PID    -   7. Vital signs before administration of study drug (temperature,        blood pressure, pulse rate, respiratory rate)

Note: All of the above must be completed before administration of studydrug.

-   -   1. Administration of study drug    -   2. Monitor vital signs 30 minutes after injection or end of        infusion    -   3. Monitor for adverse events and serious adverse events    -   4. Post-dose blood collection        -   Serum for SYNAGIS® levels immediately after completion of            infusion (IV dose groups only) and at 0.25, 0.5, 1, 4, 8,            and 12 hours after injection/end of infusion            Study Day 1: Dose 1 Pharmacokinetic Sampling            Visit 2    -   1. Post-dose blood collection for 24 hour SYNAGIS® serum level    -   2. Monitor for adverse events and serious adverse events        Study Day 2: Dose 1 Pharmacokinetic Sampling        Visit 3    -   1. Post-dose blood collection for 48 hour SYNAGIS® serum level    -   2. Monitor for adverse events and serious adverse events        Study Day 3: Dose 1 Pharmacokinetic Sampling        Visit 4    -   1. Post-dose blood collection for 72 hour SYNAGIS® serum level    -   2. Monitor for adverse events and serious adverse events        Study Day 4: Dose 1 Pharmacokinetic Sampling        Visit 5    -   1. Post-dose blood collection for 96 hour SYNAGIS® serum level    -   2. Monitor for adverse events and serious adverse events        Study Day 5: Dose 1 Pharmacokinetic Sampling        Visit 6    -   1. Post-dose blood collection for 120 hour SYNAGIS® serum level    -   2. Monitor for adverse events and serious adverse events        Study Day 7:        Visit 7    -   1. Urinalysis    -   2. Blood collection        -   Chemistry panel (AST, ALT, BUN, creatinine)        -   CBC with differential and platelet count        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS® antibody    -   3. Monitor for adverse events and serious adverse events        Study Day 14±1:        Visit 8    -   1. Blood collection        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS® antibody    -   2. Monitor for adverse events and serious adverse events        Study Day 21±1:        Visit 9    -   1. Blood collection        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS® antibody    -   2. Monitor for adverse events and serious adverse events        Study Day 30±1:        Visit 10

All volunteers

-   -   1. Blood collection        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS®antibody    -   2. Monitor for adverse events and serious adverse events        Volunteers in IM groups only    -   3. Body weight    -   4. Urine βHCG    -   5. Vital signs before administration of study drug    -   6. Administration of study drug    -   7. Monitor vital signs 30 minutes after injection        Study Day 37±1:        Visit 11

Volunteers in IM groups only

-   -   1. Urinalysis    -   2. Blood collection        -   Chemistry panel (AST, ALT, BUN, creatinine)        -   CBC with differential and platelet count        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS® antibody    -   3. Monitor for adverse events and serious adverse events        Study Day 60±2:        Visit 12    -   1. Blood collection        -   Serum for SYNAGIS® level        -   Serum for anti-SYNAGIS antibody    -   2. Monitor for serious adverse events

E. Volunteer Evaluation Methods

-   -   a. Routine Laboratory Evaluations

Routine laboratory tests during screening and during the study will beperformed at each study site's local clinical laboratory. Urinepregnancy tests during the study will be performed in the study siteusing a licensed test. Abnormal laboratory results should be repeated assoon as possible (preferably within 24–48 hours).

b. Pharmacokinetic and Immunologic Evaluations

SYNAGIS® serum concentration and anti-SYNAGIS® antibodies will bemeasured by MedImmune, Inc. by ELISA.

F. Completion of Study and Loss to Follow-up

Volunteers will be considered to have completed the study if they werefollowed up through Study Day 60. It should be specified on the casereport form whether or not the volunteer completed the study follow-upprocedures through Study Day 60. Volunteers will be consideredlost-to-follow-up only if no contact has been established by the timethe study is completed such that there is insufficient information todetermine the volunteer's status at Study Day 60. The investigatorshould document attempts to re-establish contact with missing volunteersthroughout the study period. If contact with a missing volunteer isre-established, follow-up should resume according to the protocol.

Pharmacokinetic and immunologic evaluations will be made based onSYNAGIS® serum concentration and anti-SYNAGIS® antibodies measured byELISA.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. An aqueous palivizumab formulation comprising, in an aqueous carrier:(a) at least 40 mg/ml of palivizumab, or an antigen-binding fragmentthereof; and (b) histidine, wherein said formulation is substantiallyfree of surfactants and inorganic salts.
 2. An aqueous palivizumabformulation comprising, in an aqueous carrier: (a) at least 100 mg/ml ofpalivizumab, or an antigen-binding fragment thereof; (b) histidine at aconcentration of about 20 mM to about 30 mM; and (c) glycine at aconcentration of less than 2 mM, wherein said formulation issubstantially free of surfactants, inorganic salts and other excipients.3. An aqueous palivizumab formulation comprising palivizumab or anantigen-binding fragment thereof in an aqueous carrier, said formulation(i) having been prepared by a process in which, for each step of saidprocess, said palivizumab or palivizumab-antigen-binding fragment is inan aqueous phase; and (ii) being suitable for injection in a humansubject, and wherein said palivizumab or palivizumab antigen-bindingfragment is at a concentration of at least 40 mg/ml.
 4. The formulationof claim 1 or 3, wherein said palivizumab or palivizumab antigen-bindingfragment is at a concentration of at least 50 mg/ml, at least 60 mg/ml,at least 70 mg/ml, at least 80 mg/ml or at least 90 mg/ml.
 5. Theformulation of claim 1 or 3, wherein said palivizumab or palivizumabantigen-binding fragment is at a concentration of at least 100 mg/ml. 6.The formulation of claim 1, 2 or 3, wherein said palivizumab orpalivizumab antigen-binding fragment is at a concentration of at least125 mg/ml.
 7. The formulation of claim 1, 2 or 3, wherein saidpalivizumab or palivizumab antigen-binding fragment is at aconcentration of at least 150 mg/ml.
 8. The formulation of claim 1 or 3,wherein said formulation comprises histidine is at a concentration ofabout 1 mM to about 100 mM.
 9. The formulation of claim 1 or 3, whereinsaid formulation comprises histidine is at a concentration of about 10mM to about 50 mM.
 10. The formulation of claim 1 or 3, wherein saidformulation is substantially free of other excipients.
 11. Theformulation of claim 1 or 3, wherein said formulation further comprisesan excipient other than a surfactant.
 12. The formulation of claim 9,wherein said formulation further comprises an excipient other than asurfactant.
 13. The formulation of claim 11, wherein the excipient isglycine.
 14. The formulation of claim 12, wherein the excipient isglycine.
 15. The formulation of claim 13, wherein glycine is at aconcentration of less than 150 mM, less than 100 mM or less than 50 mM.16. The formulation of claim 14, wherein glycine is at a concentrationof less than 150 mM, less than 100 mM or less than 50 mM.
 17. Theformulation of claim 13, wherein glycine is at a concentration of lessthan 3 mM or less than 2 mM.
 18. The formulation of claim 14, whereinglycine is at a concentration of less than 3 mM or less than 2 mM. 19.The formulation of claim 1 or 3, wherein said formulation has a pH ofbetween about 5.5 to about 7.0.
 20. The formulation of claim 19, whereinsaid formulation has a pH of between about 5.5 to about 6.5.
 21. Theformulation of claim 13, wherein said formulation has a pH of betweenabout 5.5 to about 7.0.
 22. The formulation of claim 14, wherein saidformulation has a pH of between about 5.5 to about 7.0.
 23. Theformulation of claim 2, wherein histidine is at a concentration of about25 mM and glycine is at a concentration of 1.6 mM.
 24. The formulationof claim 2, wherein the formulation has a pH of between about 5.5 toabout 7.0.
 25. The formulation of claim 2, wherein the formulation has apH of about 5.5 to about 6.5.
 26. The formulation of claim 2, whereinthe formulation has a pH of about 6.0.
 27. The formulation of claim 11,wherein the excipient is a saccharide.
 28. The formulation of claim 27,wherein the saccharide is sucrose.
 29. The formulation of claim 28,wherein the sucrose is at a concentration of about 1% to about 20%. 30.The formulation of claim 11, wherein the excipient is a polyol otherthan mannitol.
 31. The formulation of claim 30, wherein the polyol ispolysorbate.
 32. The formulation of claim 30, wherein the polyol isTween, which is at a concentration of about 0.001% to about 1%.
 33. Theformulation of claim 1, 2 or 3, wherein the aqueous carrier is distilledwater.
 34. The formulation of claim 26, wherein the aqueous carrier isdistilled water.
 35. The formulation of claim 1 or 2, wherein theformulation is sterile.
 36. The formulation of claim 26, wherein theformulation is sterile.
 37. The formulation of claim 1 or 2, wherein theformulation is homogenous.
 38. The formulation of claim 1, 2 or 3,wherein said palivizumab or an antigen-binding fragment thereof isstable at 40° C. for at least 100 days as determined by HPSEC.
 39. Theformulation of claim 1, 2 or 3, wherein said palivizumab or palivizumabantigen-binding fragment is stable at about ambient temperature for atleast 1 year as determined by HPSEC.
 40. The formulation of claim 1, 2or 3, wherein said palivizumab or palivizumab antigen-binding fragmentis stable at 4° C. for at least 3 years as determined by HPSEC.
 41. Theformulation of claim 1, 2 or 3, wherein less than 2% of said palivizumabor palivizumab antigen-binding fragment forms an aggregate as measuredby HPSEC.
 42. The formulation of claim 1 or 2 which has been prepared bya process in which, for each step of said process, said palivizumab orpalivizumab antigen-binding fragment is in an aqueous phase.
 43. Theformulation of claim 1 or 2 which has been prepared by a process thatdoes not have a drying step.
 44. The formulation of claim 1 or 2 whichhas been prepared by a process that does not have a lyophilization step.45. A pharmaceutical unit dosage form comprising the formulation ofclaim 1, 2 or 3, which dosage form is suitable for parenteraladministration to a human and is in a suitable container.
 46. Apharmaceutical unit dosage form comprising the formulation of claim 26,which dosage form is suitable for parenteral administration to a humanand is in a suitable container.
 47. The pharmaceutical unit dosage formof claim 45, wherein the formulation is suitable for subcutaneous,intravenous or intramuscular administration.
 48. The pharmaceutical unitdosage form of claim 46, wherein the formulation is suitable forsubcutaneous, intravenous or intramuscular administration.
 49. Apharmaceutical unit dosage form comprising the formulation of claim 1, 2or 3, which dosage form is suitable for aerosol administration to ahuman and is in a suitable container.
 50. A pharmaceutical unit dosageform comprising the formulation of claim 26, which dosage form issuitable for aerosol administration to a human and is in a suitablecontainer.
 51. A sealed container comprising the formulation of claim 1,2 or
 3. 52. A sealed container comprising the formulation of claim 26.53. A method of preventing a RSV infection or one or more symptomsthereof in a subject, said method comprising administering to thesubject a prophylactically effective amount of the formulation of claim1, 2 or
 3. 54. A method of preventing a RSV infection or one or moresymptoms thereof in a subject, said method comprising administering tothe subject a prophylactically effective amount of the formulation ofclaim
 26. 55. The method of claim 53, wherein the formulation isadministered parenterally.
 56. The method of claim 54, wherein theformulation is administered parenterally.
 57. The method of claim 53,wherein the formulation is administered intramuscularly, intravenously,subcutaneously or intranasally.
 58. The method of claim 54, wherein theformulation is administered intramuscularly, intravenously,subcutaneously or intranasally.
 59. The method of claim 53, wherein thesubject is a human.
 60. The method of claim 54, wherein the subject is ahuman.
 61. The formulation of claim 3, wherein said formulationcomprises histidine is at a concentration of about 1 mM to about 100 mMand the palivizumab or palivizumab antigen-binding fragment is at aconcentration of at least 100 mg/ml.
 62. The formulation of claim 3,wherein said formulation comprises histidine is at a concentration ofabout 10 mM to about 50 mM and the palivizumab or palivizumabantigen-binding fragment is at a concentration of at least 100 mg/ml.63. The formulation of claim 3, wherein the formulation is substantiallyfree of other excipients and the palivizumab or palivizumabantigen-binding fragment is at a concentration of at least 100 mg/ml.64. The formulation of claim 3, wherein the formulation furthercomprises an excipient other than a surfactant and the palivizumab orpalivizumab antigen-binding fragment is at a concentration of at least100 mg/ml.
 65. The formulation of claim 3, wherein said formulationcomprises histidine is at a concentration of about 10 mM to about 50 mMand the palivizumab or palivizumab antigen-binding fragment is at aconcentration of at least 100 mg/ml, and wherein said formulationfurther comprises an excipient other than a surfactant.
 66. Theformulation of claim 64, wherein the excipient is glycine.
 67. Theformulation of claim 65, wherein the excipient is glycine.
 68. Theformulation of claim 66, wherein glycine is at a concentration of lessthan 150 mM, less than 100 mM or less than 50 mM.
 69. The formulation ofclaim 67, wherein glycine is at a concentration of less than 150 mM,less than 100 mM or less than 50 mM.
 70. The formulation of claim 66,wherein glycine is at a concentration of less than 3 mM or less than 2mM.
 71. The formulation of claim 67, wherein glycine is at aconcentration of less than 3 mM or less than 2 mM.
 72. The formulationof claim 1 or 3, wherein the formulation has a pH of between about 5.5to about 7.0 and the palivizumab or palivizumab antigen-binding fragmentis at a concentration of at least 100 mg/ml.
 73. The formulation ofclaim 72, wherein said formulation has a pH of between about 5.5 toabout 6.5.
 74. The formulation of claim 66, wherein said formulation hasa pH of between about 5.5 to about 7.0.
 75. The formulation of claim 67,wherein said formulation has a pH of between about 5.5 to about 7.0. 76.The formulation of claim 23, wherein the formulation has a pH of about6.0.
 77. The formulation of claim 64, wherein the excipient is asaccharide.
 78. The formulation of claim 77, wherein the saccharide issucrose.
 79. The formulation of claim 78, wherein the sucrose is at aconcentration of about 1% to about 20%.
 80. The formulation of claim 64,wherein the excipient is a polyol other than mannitol.
 81. Theformulation of claim 80, wherein the polyol is polysorbate.
 82. Theformulation of claim 80, wherein the polyol is Tween, which is at aconcentration of about 0.001% to about 1%.
 83. The formulation of claim76, wherein the aqueous carrier is distilled water.
 84. The formulationof claim 76, wherein the formulation is sterile.
 85. A sealed containercomprising the formulation of claim
 76. 86. A method of preventing a RSVinfection or one or more symptoms thereof in a subject, said methodcomprising administering to the subject a prophylactically effectiveamount of the formulation of claim
 76. 87. The method of claim 86,wherein the formulation is administered parenterally.
 88. The method ofclaim 86, wherein the formulation is administered intramuscularly,intravenously, subcutaneously or intranasally.
 89. The method of claim86, wherein the subject is a human.